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Experiments

Experiments is the specific procedures in our Wetlab process

Protocol

In this module, we will show you in detail all the operating steps involved in our Wetlab process. You can quickly find the experimental procedure information you need through the following five sections, including "Plasmid Construction", "RNA Interference", "Delivery Systems", "Metarhizium", and "Detection Module". Additionally, you can use the brief introductions or subheadings in each section to help you locate the corresponding operating steps.

Plasmid Construction

This section covers:
the methods for constructing target plasmids using conventional molecular biology methods.

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RNA Interference

This section covers:
the methods for producing dsRNA or shRNA via in vitro transcription.

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Delivery Systems

This section covers:
Construction and characterization of MS2 VLP plasmids and Assay for the protective performance of MS2 VLP

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Metarhizium

This section covers:
Preparation of Metarhizium spore suspension and Efficacy verification of Metarhizium recombinant strains

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Detection Module

This section covers
Induction of Bacillus subtilis using sucrose and Quantification of methyl salicylate by LC-MS method

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Plasmid Construction

In this module, we constructed a total of 12 types of plasmids. You may refer to the specific experimental methods for the design and transformation of these plasmids. The corresponding function of each plasmid can be found in table 1, while their sequences can be retrieved from the Sequence module of the Experiment webpage. For more detailed information, you may also access the Results page, which allows you to gain a deeper understanding of the design rationale and design outcomes of each plasmid.

Figure 7

Transformation Method for Escherichia coli

1. Plasmid Transformation

  1. Take the competent cells out of the -80°C refrigerator, thaw them on ice, and then transfer them to a biosafety cabinet.
  2. Thaw the centrifuge tube containing 4 μg of plasmid on ice, then place it in a centrifuge and centrifuge at 5000 rpm for 1 min. After centrifugation, add 40 μL of sterile water to the tube, mix thoroughly, and adjust the plasmid concentration to 100 ng/μL.
  3. Add 10 μL of the 100 ng/μL target plasmid solution to 100 μL of competent cells, mix by gently flicking the bottom of the tube, and incubate on ice for 25 min.
  4. Place the centrifuge tube in a 42°C metal bath for heat shock treatment (45 seconds), then immediately transfer it to ice and incubate for 3 minutes.
  5. Add 700 μL of antibiotic-free LB liquid medium (prepared as shown in Table 1) to the transformed competent cells, mix well, and place the mixture in a 37°C shaker (220 rpm) for 60 minutes to recover the bacterial cells.
Figure 7

2. Sectional Streaking and Colony Verification

  1. Transfer the resuscitated competent cell suspension to a centrifuge and centrifuge at 5000 rpm for 1 min. Remove part of the supernatant, leaving approximately 100 μL of supernatant to resuspend the bacterial pellet. After suspending the bacteria, dip a small amount of the bacterial suspension with a sterile inoculating loop and perform sectional streaking on the surface of LB agar medium containing antibiotics (formulated as specified in Table 3). After streaking, invert the plate and incubate overnight at 37°C in an incubator.
  2. Add an appropriate amount of antibiotics to 5 mL of antibiotic-free LB broth to prepare antibiotic-containing LB broth. Subsequently, pick a single colony into the aforementioned medium and incubate in a 37°C orbital shaker at 220 rpm until the OD value reaches 0.8.
  3. A small amount of the bacterial culture is used for PCR reactions (the specific reaction system is shown in Table 4) to confirm that the target plasmid has been successfully transformed into Escherichia coli. The primers used were purchased from Sangon Biotech Co., Ltd., and 2×Taq Master Mix was purchased from Vazyme Biotech Co., Ltd.
Figure 7
Figure 7

Method for Transformation of Metarhizium

Amplification of Target Plasmids

  1. Retrieve DH5α competent cells from -80°C, quickly place them on ice, and let stand for 5 min to allow the cell pellet to thaw.
  2. In a laminar flow hood, pipette 5 μL (10~100 ng) of the target plasmid, add it to the competent cells, gently tap the bottom of the EP tube with fingers to mix, and incubate on ice for 25 min.
  3. Perform heat shock in a 42°C water bath for 45 seconds, then immediately return to ice and let stand for 2 min.
  4. Add 700 μL of sterile antibiotic-free LB broth to the centrifuge tube, mix well, and place in a 37°C shaker; incubate with shaking at 200 rpm for 1 h.
  5. Centrifuge at 5000 rpm for 1 min to harvest the bacterial cells, discard approximately 600 μL of the supernatant, gently resuspend the cell pellet by pipetting with the remaining LB broth, and spread onto LB agar medium containing kanamycin (knaR) at a working concentration of 50 μg/mL.
  6. Incubate in a 37°C incubator for 24 h. Pick a single colony into 500 μL of sterile water, and heat in a 100°C heat block for 10 min.
  7. Perform PCR identification according to the system (Table 3) in the instructions.
  8. Take the bacterial culture and 50% glycerol, mix them thoroughly at a 1:1 ratio, and store in a -80°C freezer.

Transformation of Vector into Agrobacterium tumefaciens AGL1

  1. Retrieve Agrobacterium tumefaciens AGL1 competent cells from -80°C, quickly place them on ice, and let stand to allow the cell pellet to thaw.
  2. In a laminar flow hood, pipette 5 μL of the vector plasmid obtained in the previous step into 50 μL of competent cells, gently tap the tube wall with fingers to mix, and incubate on ice for 5 min.
  3. Transfer the mixture to liquid nitrogen for 5 min, then incubate in a 37°C water bath for 5 min, immediately return to ice, and let stand for 5 min.
  4. Add 700 μL of sterile antibiotic-free LB broth, mix well, and place in a 28°C shaker; incubate with shaking at 200 rpm for 2–3 h.
  5. Centrifuge at 6000 rpm for 1 min to harvest the bacterial cells, discard approximately 600 μL of the supernatant, gently resuspend the cell pellet by pipetting with the remaining LB broth, and spread onto LB agar medium containing kanamycin sulfate (working concentration: 50 μg/mL).
  6. After labeling, invert the plate and incubate in a 28°C incubator for 48 h.
  7. Once single colonies grow on the plate, pick a single colony and inoculate it into fresh LB broth containing kanamycin sulfate (working concentration: 50 μg/mL). After incubating with shaking overnight, take 3 mL of the bacterial culture for PCR verification; the system and protocol refer to Step 7 in Section 6.3.
  8. Take the bacterial culture and 50% glycerol, mix them thoroughly at a 1:1 ratio, and store in a -80°C freezer.

Transformation and Screening of Metarhizium

Transformation of Metarhizium by Agrobacterium-mediated transformation method

  1. After verification of Agrobacterium, take an appropriate amount of the bacterial culture and add it to LB broth containing kanamycin (working concentration: 50 μg/mL). Incubate in a 28°C shaker at 200 rpm for 12 h.
  2. Collect 6 mL of the bacterial culture and centrifuge at 4000 rpm for 10 min at 4°C. Discard the supernatant, resuspend the cell pellet with 1 mL of IM liquid medium, transfer to a 50 mL centrifuge tube, then add 20 mL of IM liquid medium (prepared as shown in Table 5-2). Adjust the concentration to an OD660 of approximately 0.2.
  3. Wrap the centrifuge tube with paper, place it in a shaker at 28°C, and incubate with 200 rpm for 6 hours until the OD600 reaches approximately 0.6.
  4. Take 200 μL of the aforementioned bacterial suspension and mix it thoroughly with 200 μL of spore suspension (concentration: 1×106 spores/mL).
  5. Take 50 μL of the mixed solution and spread it evenly on IM solid medium (prepared as shown in Table 5-5) with a sterile filter membrane placed on top. Incubate in a 28°C incubator in the dark for 48 hours.
Figure 7
  1. Remove the filter membrane from the IM solid medium and place it on m-100 solid medium (prepared as shown in Table 5-9).
Figure 7
  1. The spore suspension is prepared and then spread separately onto PDA and SDAY (prepared as shown in Table 5-6) media containing glufosinate-ammonium and cephalosporin for further cultivation.

Verification of Recombinant Metarhizium Strains

Genomic DNA Extraction of Recombinant Metarhizium Strains

  1. When the mycelia grow to half the area of the PDA Petri dish, scrape the mycelia into 1.5 mL microcentrifuge tubes using pipette tips.
  2. Evaporate the moisture from the microcentrifuge tubes in a 60°C heat block. After quick-freezing with liquid nitrogen, grind the mycelia into a powder using a grinding rod.
  3. Add 400 μL of Buffer Digestion and 4 μL of β-mercaptoethanol, then vortex to mix thoroughly.
  4. Transfer the tubes to a 65°C water bath and incubate for 1 h until the cells are completely lysed. During the water bath incubation, invert the tubes to mix once every 10 min.
  5. After the water bath, add 200 μL of Buffer PF, invert thoroughly to mix, and then place the tubes in a -20°C freezer for 5 min.
  6. After the cold treatment, centrifuge at 10,000 rpm for 5 min at room temperature. Transfer the clear supernatant to a new 1.5 mL microcentrifuge tube.
  7. Add 600 μL of isopropanol to the supernatant, invert approximately 8 times to mix well, and let stand at room temperature for 3 min. Subsequently, centrifuge at 10,000 rpm for 5 min at room temperature and discard the supernatant.
  8. Add 1 mL of 75% ethanol to the pellet, invert to rinse for 2 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant.
  9. Repeat Step 8 once.
  10. Open the tube lid and invert the tube at room temperature for 10 min until the residual ethanol is completely evaporated.
  11. Dissolve the obtained DNA in 60 μL of TE Buffer. The extracted DNA is stored at -20°C and can be used for subsequent PCR identification and restriction enzyme digestion identification.

Genomic PCR Verification

Perform PCR identification in accordance with the following system and protocol (Table 6).

Figure 7

RNA Interference Verification

This section outlines the complete workflow for RNA interference verification, including plasmid extraction, dsRNA and shRNA synthesis, and functional validation through aphid gene silencing experiments. The protocols detail the preparation of linear templates, transcription reactions, and quantitative analysis of gene silencing efficiency.

Plasmid Extraction

Extract plasmids using the TIANGEN Plasmid Mini Extraction Kit.

  1. Add 500 μL of Equilibration Buffer BL to Adsorption Column CP3 (place the adsorption column into a collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
  2. Take 1–5 mL of overnight-cultured bacterial culture, add it to a centrifuge tube, centrifuge at 12,000 rpm for 1 min using a conventional benchtop centrifuge, and aspirate the supernatant as much as possible.
  3. Add 250 μL of Solution P1 (RNaseA should be added first) to the centrifuge tube with the bacterial pellet remaining, and thoroughly resuspend the bacterial pellet using a pipette or vortex mixer.
  4. Add 250 μL of Solution P2 to the centrifuge tube, gently invert it up and down 6–8 times to fully lyse the bacteria.
  5. Add 350 μL of Solution P3 to the centrifuge tube, immediately invert gently up and down 6–8 times to mix thoroughly; white flocculent precipitates will appear at this time. Then centrifuge at 12,000 rpm for 10 min.
  6. Transfer the supernatant collected in the previous step to Adsorption Column CP3 (place the adsorption column into a collection tube) using a pipette, taking care to avoid aspirating the precipitate. Centrifuge at 12,000 rpm for 30–60 sec, discard the waste liquid in the collection tube, and place Adsorption Column CP3 back into the collection tube.
  7. Add 600 μL of Wash Buffer PW (absolute ethanol should be added first) to Adsorption Column CP3, centrifuge at 12,000 rpm for 30–60 sec, discard the waste liquid in the collection tube, and place Adsorption Column CP3 back into the collection tube.
  8. Repeat the previous step.
  9. Place Adsorption Column CP3 into the collection tube, centrifuge at 12,000 rpm for 2 min to remove residual wash buffer in the adsorption column. Open the lid of Adsorption Column CP3 and place it at room temperature for 20 min to completely air-dry residual wash buffer in the adsorption material.
  10. Place Adsorption Column CP3 into a clean centrifuge tube, add 60 μL of RNase-free water to the middle of the adsorption membrane, let stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.
  11. Use 1 μL of the sample for Nanodrop analysis to preliminarily determine the presence of nucleic acids in the eluate, with RNase-free water as the blank control.
  12. Prepare the double enzyme digestion system according to Table 1 below, gently mix each component with a pipette, collect the mixture by brief centrifugation in a palm centrifuge, and incubate at 37°C for 30 min.
  13. Prepare a 2% (w/v) agarose gel: weigh 0.6 g of agarose, add 30 mL of 1× TAE, heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately 60°C, add SuperRed dye to a final concentration of 0.01% (v/v) (3 μL). Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
  14. Prepare the loading sample: mix 10 μL of the incubated sample with 2 μL of 6× Loading Buffer (purchased from Bioshop), and place the mixture on ice.
  15. Electrophoresis: Remove the comb from the solidified agarose gel, place the gel into the electrophoresis tank with the loading wells facing the negative electrode, and add TAE electrophoresis buffer. Load 10 μL of 1000 bp DNA marker and 10 μL of 10000 bp marker into the loading wells, respectively, and load 12 μL of each sample. Run at 150 V for 25 min.

dsRNA Synthesis

Preparation of Linear Templates

  1. Prepare the single/double restriction enzyme digestion system (as shown in Table Table 7-1) (depending on the restriction enzyme sites set for different templates). Gently mix each component with a pipette, collect by brief centrifugation, and incubate at 37°C for 30 min. All restriction enzyme sites of the templates used are set as shown in Table 7-2.
Restriction enzyme digestion system table
  1. Incubate the prepared reaction system at 37°C for 2–4 hours. After the reaction, incubate at 72°C for 10 min, then cool at room temperature for 20 min for annealing.
  2. After the reaction, white precipitates may appear; these are magnesium pyrophosphate formed by free pyrophosphate and magnesium ions during the reaction, which will not affect subsequent experiments. You can choose to add some EDTA to remove it, or centrifuge at 2500×g for 1 min using a benchtop centrifuge to recover the supernatant.
  3. Add 3 μL of DNase I (1 U/μL) and 1 μL of RNase T1 to the reaction system, incubate at 37°C for 15 min to digest the linear templates used for in vitro transcription and partial single strands of the RNA product.
  4. Prepare a 2% (w/v) RNase-free agarose gel (selected according to the band length): Weigh 0.6 g of agarose, add 30 mL of 1× RNase-free TAE, heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately 60°C, add 3 μL of SuperRed dye. Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
  5. Prepare the loading sample: Mix 1 μL of sample, 9 μL of RNase-free water, and 2 μL of 6× Loading Buffer, then place the mixture on ice.
  6. Electrophoresis: Remove the comb from the solidified agarose gel, then place the gel into the electrophoresis tank. An ice-water bath is used outside the electrophoresis tank to reduce heat generation during gel running. Orient the loading wells toward the negative electrode, and add 1× RNase-free TAE electrophoresis buffer until the gel is submerged by approximately 1 mm. Load 10 μL of 500 bp DNA marker into the loading well, and load 12 μL of each sample. Run at 180 V for 25 min.
  7. Dilute each RNA sample with RNase-free water to an appropriate dilution factor, then measure the concentration using a Nanodrop, with RNase-free water as the blank control.

shRNA Synthesis

Preparation of Linear Templates

  1. Prepare a 50 μL restriction enzyme digestion system according to the following system (Table 8).
Restriction enzyme digestion system for shRNA
  1. Incubate the system in a 42°C heat block for 30 minutes.
  2. Perform gel electrophoresis using a 2% 1×TAE gel to verify the correctness of the linear template. Proceed to the next step if the length is correct.

Transcription Reaction

  1. Use the BELONGBIO T7 Transcription Kit Plus to transcribe the linear template.
  2. As shown in the table below (Table 9), prepare a 20 μL transcription system in a 1.5 mL microcentrifuge tube:
Transcription system composition
  1. Incubate the prepared transcription system in a heat block at 37°C for 4 hours. After 4 hours, adjust the heat block to 72°C and incubate for 10 minutes, then remove the tube.
  2. Once the microcentrifuge tube has cooled to room temperature, add 3 μL of DNaseⅠ to each system, and continue incubating at 37°C for 15 minutes. Store the resulting product at -80°C.
  3. Prepare a 2% (w/v) RNase-free agarose gel (selected according to the band length): Weigh 0.6 g of agarose, add 30 mL of 1× RNase-free TAE, and heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately 60°C, add 3 μL of SuperRed dye. Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
  4. Prepare the loading sample: Mix 1 μL of sample, 9 μL of RNase-free water, and 2 μL of 6× loading buffer, then place the mixture on ice.
  5. Electrophoresis: Remove the comb from the solidified agarose gel, then place the gel into the electrophoresis tank. Use an ice-water bath outside the electrophoresis tank to reduce heat buildup during gel running. Orient the loading wells toward the negative electrode, and add 1× RNase-free TAE electrophoresis buffer until the gel is submerged by approximately 1 mm. Load 10 μL of 500 bp DNA marker into the loading well, and load 12 μL of each sample. Run at 180 V for 25 minutes.
  6. Dilute each RNA sample with RNase-free water to an appropriate dilution factor, then measure the concentration using a Nanodrop, with RNase-free water as the blank control.

RNA Interference of Aphid Genes

Verification of Aphid Gene Silencing by RNA-Containing Diet

Preparation of Stock Solutions for Each Component

  1. Amino acid stock solution (10×): Weigh 20 g of arginine, 10 g of threonine, 10 g of leucine, 6 g of tryptophan, 30 g of aspartic acid, 5 g of glycine, 1 g of tyrosine, 1 g of γ-aminobutyric acid (GABA), 10 g of histidine, 10 g of lysine, 6 g of phenylalanine, 5.5 g of asparagine, 30 g of glutamine, 5 g of serine, 2.5 g of cysteine, 6 g of methionine, 10 g of valine, 7.5 g of isoleucine, 10 g of glutamic acid, 5 g of alanine, 5 g of proline, and 0.5 g of cystine into a 1 L conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
  2. Vitamin stock solution (5×): Weigh 2.5 g of ascorbic acid, 0.125 g of pyridoxine hydrochloride, 0.25 g of calcium pantothenate, 2.5 g of choline chloride, 0.125 g of riboflavin, 0.5 g of nicotinic acid, 0.01 g of biotin, 0.5 g of p-aminobenzoic acid, 0.125 g of thiamine hydrochloride, 0.025 g of folic acid, and 2.5 g of inositol into a 1 L conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
  3. Salt and trace element stock solution (2×): Weigh 10 g of potassium dihydrogen phosphate, 0.014 g of calcium dihydrogen phosphate, 0.004 g of ferric citrate, 0.014 g of magnesium sulfate, 0.006 g of copper chloride, 0.026 g of zinc chloride, 4 g of magnesium chloride, 0.034 g of calcium lactate, 0.01 g of sodium dihydrogen phosphate, 0.006 g of sodium chloride, and 0.01 g of manganese chloride into a 1 L conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
  4. Sugar stock solution (10×): Weigh 300 g of sucrose into a 100 mL conical flask. Add HPLC-grade ultrapure water to dissolve, and while stirring, add water to reach 100 mL. Once the sucrose is completely dissolved with no particles, aliquot into reagent bottles for later use.

Operation for Nutrient Solution Mixing (100 mL)

Take a 100 mL conical flask, and sequentially add 10 mL of amino acid stock solution (10×), 20 mL of vitamin stock solution (5×), 50 mL of salt and trace element stock solution (2×), and 10 mL of sugar stock solution (10×). After mixing, add HPLC-grade ultrapure water to the 100 mL mark.

Preparation and Addition of Citrus Juice

Take 5 fresh tender citrus leaves, sample each leaf using a 1 cm diameter punch. Mix the citrus leaf samples, add 5 mL of ultrapure water, and grind in a sterilized mortar. Centrifuge and collect the supernatant for later use. Add 4 mL of this supernatant to the prepared 100 mL nutrient solution.

pH Adjustment and Sterilization

Immerse the pH meter probe into the solution to monitor the pH value (measure after stirring uniformly). Use a pipette to add 1% NaOH or HCl solution dropwise to adjust the pH to 7.4–7.7.

Sterilize using a 0.22 μm microporous filter membrane and matching filtration device. Aliquot filtrate into sterile reagent bottles and store at -20°C for later use.

Nutrient solution mixing table

Diet Rearing

The aphid rearing method refers to the protocol described by Li et al. (1997), with specific operations as follows: The aphid rearing chambers use open-ended polymethyl methacrylate (PMMA) tubes with a diameter of 3.2 cm and a height of 3 cm. Cut Parafilm into 1.5 cm² pieces, stretch each piece by hand to cover one end of a glass tube, and irradiate it under ultraviolet (UV) light for 15 minutes. After that, add 150 μL of artificial nutrient solution to the surface of the Parafilm, and then cover it with two additional layers of Parafilm. Place the aphids into the tube, and repeat the same operation for the other end of the tube. Rear the aphids in an illuminated incubator under the following conditions: temperature of 24°C ± 1°C, relative humidity of 85%–90%, and photoperiod of 16:8 (L:D, Light:Dark).

The aphid source is collected from field crops; aphids of the same instar or from the same brood of viviparous aphids are selected as the test population. After being starved for approximately 3 hours, the aphids are transferred into the glass tubes, with 200 aphids per tube.

Plant-Mediated RNA Interference

  1. Take the young instar aphids required for the experiment, 30 aphids per group, and starve them for 3 hours for later use.
  2. Cut tender citrus stems of approximately 10 cm in length, soak them in 75% ethanol for sterilization, and set aside.
  3. Prepare the required RNA concentration using RNase-free water (the concentration can be calibrated by Nanodrop), and take an equal volume of RNase-free water as the control for later use.
  4. Prepare the rearing devices needed for the experiment: Take an appropriate number of 1.5 mL and 50 mL microcentrifuge tubes. Make a neat cut along the bottom edge of the 1.5 mL microcentrifuge tube, ensuring the cut is as even as possible during operation.
  5. After labeling the group numbers, add 300 μL of RNase-free water to the control group. Insert the 10 cm citrus tender stem into the bottom-cut 1.5 mL microcentrifuge tube, and tightly wrap the tube mouth with Parafilm to prevent aphids from accidentally falling in. For the experimental group, replace RNase-free water with the prepared RNA, repeat the above operations until all groups are set up. Place them in a laminar flow hood and let stand for 3 hours to allow the plants to absorb the liquid.
  6. Gently transfer the aphids to the 10 cm citrus tender stems using a writing brush, 30 aphids per group.
  7. Unscrew the lid of the 50 mL centrifuge tube, and use non-toxic glue to vertically fix the 1.5 mL microcentrifuge tube (with the open end facing up) containing the inserted citrus tender stem at the center of the lid. After the glue solidifies, slowly fit the 50 mL centrifuge tube wall over the lid and tighten the lid. Wrap the end of the 50 mL centrifuge tube away from the lid with gauze, then transfer it to an artificial climate incubator for cultivation. The cultivation conditions are strictly set as follows: temperature 25±1°C, relative humidity 70±5%, photoperiod 16:8 hours (Light:Dark).
  8. Observe the growth status of aphids and tender stems every 12 hours, and record the number of dead aphids.
  9. Rearing maintenance: Replace the rearing devices every two days. In the laminar flow hood, collect dead aphids into new 1.5 mL microcentrifuge tubes, flash-freeze them in liquid nitrogen, and store them in a -80°C refrigerator for later use. Gently transfer surviving aphids with a writing brush to new microcentrifuge tubes containing freshly sterilized citrus tender stems (new tubes prepared as per the aforementioned steps) to ensure sustained nutrient supply.
  10. After 5 days of observation, collect all remaining aphids into the 1.5 mL microcentrifuge tubes containing dead aphids as mentioned above, clearly label the group information, flash-freeze them in liquid nitrogen, and store them in a -80°C refrigerator for later use.

Calculation of RNA Lethal Efficiency

Aphids are marked as dead if they fall off the plant and lie on their side, show no antenna movement when stimulated with a writing brush, or have a withered body. After recording the mortality data of the control group and experimental group, correct the mortality data according to Abbott's formula.

The specific formula is as follows:

Abbott's formula for mortality correction

Figure.01

Quantitative Detection of mRNA Silencing Efficiency of Target Genes

Method Using Aphid Total RNA Extraction Kit

Preparations Before RNA Extraction Experiment

  1. Wear a mask and gloves;
  2. Environmental treatment: Spray RNA Eraser on all surfaces that may be contacted first, then wipe dry with ethanol;
  3. Unseal new pipette tips to be used; (For detailed procedures, refer to Invitrogen's PureLink™ RNA Mini Kit)

RNA Extraction Procedure

  1. Take out the experimental aphid samples previously stored in a -80°C refrigerator. Grind the aphids with a manual grinding rod, then add 600 μL of Lysis Buffer and 6 μL of β-mercaptoethanol. Centrifuge at 12,000×g for 2 min at 25°C (room temperature). Transfer the supernatant to a new collection tube and discard the insect cuticle precipitate.
  2. Add an equal volume (600 μL) of 70% ethanol to the collection tube containing the supernatant lysis buffer. If precipitates are visible, gently disperse them.
  3. Transfer up to 700 μL of the sample into a spin column (with a collection tube) at a time, and centrifuge at 12,000×g for 15 seconds. Discard the flow-through, then reinsert the spin column back into the same collection tube.
  4. Repeat the previous step to transfer the remaining sample into the same collection tube until all the sample is transferred.
  5. Add 700 μL of Wash Buffer Ⅰ to the spin column, and centrifuge at 12,000×g for 15 seconds at room temperature. Discard the flow-through and the collection tube, then place the spin column into a new collection tube.
  6. Add 500 μL of Wash Buffer Ⅱ to the spin column, and centrifuge at 12,000×g for 15 seconds at room temperature. Discard the flow-through, and keep the spin column in the same collection tube.
  7. Repeat the previous step once.
  8. Centrifuge the empty spin column (with the collection tube) at 12,000×g for 1 min at room temperature.
  9. Add 30–50 μL of Elution Buffer to the spin column.
  10. After standing at room temperature for 1 min, centrifuge at 12,000×g for 2 min at room temperature.
  11. Repeat the previous step 2–3 times using the collected eluate.
  12. Measure the concentration of the extracted RNA using a Nanodrop; quantify the RNA by measuring the absorbance at 260 nm. Evaluate the purity of all RNA samples by the absorbance ratios of optical density (OD) 260/280 and OD 260/230.
  13. Confirm the integrity of the RNA by 1% agarose gel electrophoresis.
  14. Store the RNA samples at -80°C for later use or proceed directly to reverse transcription.

cDNA Synthesis

Please refer to the kit: TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGene)

Reaction System:

cDNA synthesis reaction system

Reaction Process:

cDNA synthesis reaction process

qRT-PCR

1. Reference Gene Selection & Primer Design

Reference genes and primer sequences

2. Setup of the qPCR System

Configure qPCR system:

qPCR reaction system

Paving of a 96-well plate:

96-well plate layout

3. Running the qPCR Program

qPCR thermal cycling program

4. Data Processing

Calculations were performed using relative quantification (ΔΔCt method).

All measured values are referenced to the value of 1.0 obtained from the RNase-free water (control group) treatment. Error bars represent the standard deviation (SD) derived from at least three biological replicates. Statistical analysis of all datasets was conducted using a one-tailed unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Characterization and Purification of Proteins

Bacterial Lysis

  1. Centrifuge the induced bacterial culture at 7,000 rpm for 5 min at 4°C, and discard the supernatant. Then resuspend the bacterial pellet in 1× Phosphate-buffered Saline (PBS) solution, centrifuge again, and discard the supernatant.
  2. Weigh the empty centrifuge tube. Add non-denaturing lysis buffer (300 mM sodium chloride, 50 mM sodium dihydrogen phosphate) at a ratio of "4 mL per gram of wet bacterial weight", and add Protease Inhibitor Cocktail, Universal, 100× to the lysis buffer, then resuspend the bacteria.
  3. Add lysozyme to the bacterial resuspension to a final concentration of 1 mg/mL, mix gently, and incubate the centrifuge tube on ice for 30 min.
  4. Keep the treated bacterial solution on ice and lyse using an ultrasonic cell disruptor (Xinzhi). Set the ultrasonic power to 200–300 W, with an ultrasonic mode of "6 seconds on, 9 seconds off", and a total sonication time of 35 minutes. Maintain an ice bath during lysis to prevent target protein denaturation due to increased temperature of the bacterial solution.
  5. After sonication, add an equal volume of nuclease-free water to the bacterial solution to dilute the cation concentration in the system. Then add Benzonase Nuclease to a final concentration of 100 U/mL. Simultaneously, add MgCl₂ to achieve a final concentration of 2 mM Mg²⁺ in the system to activate the benzonase nuclease, then incubate the bacterial solution at 37°C for 45 min.
  6. Centrifuge the bacterial solution treated with Benzonase Nuclease at 10,000×g for 20 min at 4°C, collect the supernatant containing the target protein, and filter the supernatant through a 0.22 μm polyethersulfone (PES) syringe filter to remove unbroken bacteria and cell debris. Collect the filtered supernatant.

Protein Purification

Purify the protein using Beyotime's His-tag Protein Purification Kit (Reduction-Resistant Chelating Type):

  1. According to the ratio of "4 mL bacterial lysate supernatant per 0.5 mL resin" (equivalent to 1 mL of 50% resin suspension) (1:8), take an appropriate amount of well-mixed 50% BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type), centrifuge at 1,000×g for 10 seconds at 4°C, and discard the resin storage solution. Add 1 column volume (CV) of non-denaturing lysis buffer to the resin, mix gently to equilibrate the resin, centrifuge again at 1,000×g for 10 seconds at 4°C to discard the liquid, and repeat this equilibration step 1–2 times. Finally, discard the equilibration buffer.
  2. Fully mix the equilibrated BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type) with the bacterial lysate supernatant, and incubate on a 4°C orbital shaker (80 rpm) with gentle shaking for 60 minutes.
  3. Load the mixture of resin and lysate supernatant into an empty 12 mL affinity chromatography column tube, open the bottom cap of the purification column, and allow the liquid to flow out naturally under gravity. During this process, collect approximately 20 μL of the flow-through for subsequent analysis of the binding efficiency between the protein and the resin.
  4. Perform 8 column washing steps: Add 1 CV of non-denaturing washing buffer to the column each time. After the liquid flows out completely, collect 20 μL of the washing solution for subsequent analysis of protein elution efficiency.
  5. After column washing, perform 9 elution steps for the target protein: Add 1 CV of non-denaturing elution buffer to the column each time. After the liquid flows out naturally, collect each eluate into pre-labeled centrifuge tubes for subsequent experiments.
  6. Take 80 μL of the sample, add 20 μL of 5× SDS-PAGE Sample Loading Buffer, and vortex for 10 seconds. Boil in a 100°C heat block for 5 min (to promote full protein denaturation), then immediately transfer to ice to cool for 3 min (to terminate the denaturation reaction). After cooling, centrifuge the sample at 12,000×g for 1 min, and load the supernatant onto a 12% polyacrylamide separating gel for SDS-PAGE electrophoresis. After electrophoresis, stain with Coomassie Brilliant Blue R-250 (solution system as shown in Table 17; Table 18) and destain until bands are clear. Based on the electrophoresis results, select the original samples corresponding to lanes with clear target bands and minimal interference from contaminating bands for subsequent experimental analysis.
Table 17
Table 18

Assembly and Characterization of VLP Nanoparticles

Acid Hydrolysis

  1. At a ratio of "target protein to glacial acetic acid of 1:2", add twice the volume of glacial acetic acid to the concentrated eluate, and incubate on ice for 30 minutes to denature the capsid, decompose it into protein dimers, and precipitate RNA. Subsequently, centrifuge at 10,000×g for 20 minutes at 4°C to remove the precipitated nucleic acids.
  2. Appropriately dilute the supernatant with ultrapure water and transfer it to an Amicon® Ultra centrifugal ultrafiltration tube (Specification: 10 kDa molecular weight cut-off (MWCO), sample volume 2 mL, regenerated cellulose membrane). Filter with assembly buffer (42 mM Tris, pH 7.5; 84 mM NaCl; 3 mM acetic acid; 1 mM EDTA, pH 8.0) for 5 times to remove high-concentration glacial acetic acid, and collect the concentrated sample via reverse centrifugation.
  3. According to the manufacturer's instructions, quickly determine the content of the target protein in the solution using the Bradford method.

In Vitro Assembly

  1. Clean 1.5 mL microcentrifuge tubes with RNaseEraser (Forgene), and after air-drying, prepare the assembly system according to Table 19. Mix all components thoroughly, then incubate at 21°C for 10 minutes.
  2. Add 1 μL of RNase A to the sample to a final concentration of 100 μg/mL, incubate for digestion for 30 minutes. Then add 3 μL of RiboLock RNase Inhibitor (40 U/μL) at a ratio of "RNase inhibitor to reaction system of 3/100 (v/v)" to terminate the enzymatic hydrolysis reaction of RNase A.
Table 19

Reverse Transcription PCR (RT-PCR)

Perform RT-PCR using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix:

  1. Pipette 2 μL of 0.1 μg/μL Random Primer (N9) and 14 μL of assembled VLP nanoparticles into the same 0.2 mL flat-cap thin-walled tube. Mix thoroughly, incubate in a 65°C heat block for 5 min, then place on ice for 2 min.
  2. Add 20 μL of 2× TS Reaction Mix, 2 μL of RT/RI Enzyme Mix, and 2 μL of gDNA Remover to the above-treated solution. Mix thoroughly, then place in a Biometra TAdvanced instrument. Set the PCR program as: "incubate at 25°C for 10 min, then at 42°C for 50 min".

Verification by Agarose Gel Electrophoresis

  1. Prepare a 1% (w/v) agarose gel: Weigh 0.6 g of agarose, add 60 mL of assembly buffer (Table 19) prepared with RNase-free water, and heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately 60°C, add 3 μL of SuperRed dye to a final concentration of 0.01% (v/v). Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
  2. After the gel solidifies, place it in an electrophoresis tank filled with assembly buffer (42 mM Tris, pH 7.5; 84 mM NaCl; 3 mM acetic acid; 1 mM EDTA, pH 8.0). Pipette 40 μL of the assembled sample into a new 1.5 mL microcentrifuge tube, then add 8 μL of 6× loading buffer. Mix thoroughly and load the entire mixture into a gel well. Load 10 μL each of DL1000 marker and DL500 marker. Run electrophoresis at 100 V for 1 hour.
  3. After electrophoresis, use a UVP Benchtop UV Transilluminator (Analytik Jena GmbH + Co. KG) to verify the presence of RNA in the VLP nanoparticles.
  4. Stain with Coomassie Brilliant Blue (prepared as shown in Table 17) for 1.5 hours, then destain with destaining solution (prepared as shown in Table 18). Repeat destaining until the background is clear.

Verification by Transmission Electron Microscopy (TEM)

  1. Sample adsorption: In a laminar flow hood, pipette 30 μL of the sample solution onto the surface of clean Parafilm. Take a 200-mesh carbon-coated copper grid, place it gently on the sample droplet with the front side (support film side) facing down, and incubate at room temperature for 10 min to ensure the sample is fully attached to the grid surface.
  2. Sample drying: Gently touch the edge of the copper grid with the edge of clean filter paper to absorb excess sample solution. Then place the copper grid with the front side facing up in a room-temperature, light-protected environment to air-dry naturally (approximately 30–60 min). Avoid air disturbance during this period to prevent contamination.
  3. Washing and negative staining: When the grid surface is in a semi-dry state, immediately place it gently on a 30 μL ultrapure water droplet with the front side facing down. Absorb excess water using filter paper as described above. Then, transfer the grid (front side down) to a 30 μL droplet of 2% phosphotungstic acid hydrate solution (pH 6.5–7.0) and stain at room temperature for 1 min. After staining, absorb excess stain with filter paper, transfer the grid to a new 30 μL ultrapure water droplet for 30 s (to remove excess stain), and finally blot dry residual water with filter paper.
  4. TEM observation: Place the prepared copper grid on the TEM sample stage, observe under an accelerating voltage of 80 kV. Adjust the magnification according to the morphological characteristics of the sample, capture clear images, and record relevant parameters.

Red Ink Immersion Experiment

  1. Select stems with similar morphology and consistent physiological status, then cut stem segments of equal length with the same number of leaves. Immerse the segments in a 15 mL EP tube containing 9 mL of red ink, and conduct time and concentration gradient experiments.
  2. After immersion, peel off the bark of the stems to expose the white phloem, and observe the diffusion of red ink.
  3. Make a cross-section at the mid-height of the stem segment, take a photo of the cross-section, and use ImageJ to measure the ratio of the red ink area to the total cross-sectional area for subsequent flow rate calculation.

Injection Experiment of Red Ink and Protein Mixture

  • Select citrus seedlings for stem injection. Use an electric drill and a matching drill bit to drill a hole in the citrus seedling; the depth of the hole should not exceed half the diameter of the trunk.
  • Inject 1 mL of the mixed solution of red ink and VLP nanoparticles (protein concentration: 50 μM) into a 5 mL trunk syringe, then add a driving piston. Next, inject 2–3 mL of clean water into the syringe to form a hydraulic driving chamber between the water and the original piston. Use the self-weight of the clean water as a stable external pressure to achieve continuous infusion of the mixed solution.
  • Insert the modified syringe mentioned above into the pre-drilled guide hole at a 45° downward angle, and leave the syringe in the trunk until red color appears in the leaves (Figure 02).
Figure 02

Figure 02

  • Cut approximately 1–1.5 g of tender stems with new leaves at different heights, grind them thoroughly in liquid nitrogen, and collect the powder.
  • Add 800 μL of non-denaturing lysis buffer (300 mM sodium chloride, 50 mM sodium dihydrogen phosphate) and Protease Inhibitor Cocktail, Universal, 100× to the lysis buffer, then vortex to mix thoroughly.
  • Centrifuge at 10,000×g for 10 minutes at 4°C to separate the supernatant.
  • Take 80 μL of the sample, add 20 μL of 5× SDS-PAGE Sample Loading Buffer, and vortex for 10 seconds. Boil in a 100°C heat block for 5 minutes (to promote full protein denaturation), then immediately transfer to ice to cool for 3 minutes (to terminate the denaturation reaction). After cooling, centrifuge the sample at 12,000×g for 1 minute, and load the supernatant onto a 12% polyacrylamide separating gel for SDS-PAGE separation.
  • Take a nitrocellulose (NC) membrane of the same size as the SDS-PAGE gel, directly place it in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to equilibrate for 10 minutes. Meanwhile, immerse 6–8 layers of qualitative filter paper in transfer buffer until fully soaked. After SDS-PAGE, carefully peel off the gel, cut off unnecessary edges (retain the target protein region), and place it in transfer buffer to equilibrate for 5 minutes.
  • Transfer the proteins from the gel to the NC membrane using a GenScript rapid wet transfer apparatus.
  • After transfer, transfer the NC membrane to TBST blocking solution containing 5% BSA, ensuring the membrane is completely submerged. Block overnight with gentle shaking on a room-temperature shaker (80 rpm).
  • Discard the blocking solution, and quickly rinse the membrane once with TBST (for 1 minute). Dilute the anti-His tag primary antibody according to the antibody instructions (1:1000, diluted in TBST containing 5% BSA), place the membrane in the primary antibody solution, and incubate for 2 hours on a room-temperature shaker (80 rpm).
  • Recover the primary antibody, transfer the membrane to TBST washing solution, and wash for 15 minutes on a room-temperature shaker (80 rpm); repeat this washing step three times.
  • Dilute the secondary antibody according to the antibody instructions (1:5000, diluted in TBST containing 5% BSA), place the membrane in the secondary antibody solution, and incubate for 2 hours on a room-temperature shaker (80 rpm).
  • Recover the secondary antibody, transfer the membrane to TBST washing solution, and wash for 15 minutes on a room-temperature shaker (80 rpm); repeat this washing step three times.
  • Mix ECL chemiluminescent substrate Solution A and Solution B at a 1:1 ratio, evenly add the mixture to the surface of the NC membrane (after blotting residual liquid with lens cleaning paper) to cover the target band region, and let stand in the dark for 1–2 minutes. Place the membrane in a chemiluminescence imager, adjust the exposure time (10 seconds – 5 minutes) according to signal intensity, capture images, and record the target band corresponding to the His-tagged protein (compare the molecular weight with the Marker).

Verification of RNA Stability Protected by MS2

  1. Prepare the Benzonase Nuclease digestion reaction system (volume 100 μL) according to Table 20, and react for 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 120 min respectively.
  2. After the reaction time elapses, add 1 μL of 50 mM EDTA to inhibit the activity of Benzonase Nuclease. Then verify the RNA in the above reaction products by agarose gel electrophoresis, and visualize protein bands using Coomassie Brilliant Blue staining solution.
Table 20

Functional Verification of Localization Peptide GBP3.1 and Cell-Penetrating Peptide TAT

Feeding Assay

  1. Resuspend 5 µg of the peptide-EGFP fusion protein in 200 µL of aphid nutrient solution.
  2. Add 100 µL of the aforementioned protein-nutrient solution using the double-membrane method.
  3. Feed 20 aphids per membrane for 24 hours, with three replicates per treatment.

Observation of Living Aphids

Pick the fed aphids with a writing brush and place them on a glass slide. Drop absolute ethanol vertically onto the aphids for anesthesia, and let stand for 1 minute to allow excess absolute ethanol to evaporate. Then, observe the prepared slide using a Zeiss Axioplan II fluorescence microscope with a FITC filter.

Observation of Frozen Sections

  • Antigen retrieval: Antigen retrieval is not routinely performed for frozen sections; it can be selectively conducted under special circumstances.
  • Circle marking: Mark a circle on the section using an immunohistochemical pen, then place the section in TBST.
  • Membrane permeabilization: Permeabilize the membrane with 0.1% Triton® X-100 for 15 minutes. Wash 3 times with TBST, 5 minutes each time.
  • Blocking: Add 10% goat serum dropwise, and incubate at 37°C for 30 minutes.
  • Primary antibody incubation: Flick off the serum, dilute the EGFP primary antibody stock solution with TBST to prepare a primary antibody working solution at a 1:500 dilution. Mix thoroughly using a vortex mixer, then add 50–100 μL of the primary antibody working solution to each section (depending on tissue size). Incubate at 4°C over two nights.
  • Secondary antibody incubation: On the third day, take the sections out of the refrigerator and let them stand at room temperature for 15 minutes to rewarm. Rinse 3 times with TBST, 3 minutes each time. Dilute the secondary antibody stock solution with TBST to prepare a secondary antibody working solution at a 1:400 dilution. Add 50–100 μL of the secondary antibody working solution to each section (depending on tissue size), and incubate at 37°C for 45 minutes. Rinse 3 times with TBST, 3 minutes each time.
  • Nuclear staining: Remove TBST, add 50–100 μL of DAPI working solution (prepared by diluting DAPI stock solution at 1:500) to each section (depending on tissue size). Stain nuclei in the dark for 5 minutes, then rinse with TBST.
  • Mounting: Mount the sections with fluorescent mounting medium, and store at 4°C in the dark.
  • Microscopic examination: Examine the sections under a microscope, and perform image acquisition and analysis.

Culture of Metarhizium

Colony Morphology

Metarhizium is a small filamentous fungus. On comprehensive Potato Dextrose Agar (PDA) medium, its colonies are distinct: initially white with dense and vigorous hyphae, later producing pale yellow-green spores that spread toward the edge of the petri dish. The reverse side of the medium appears yellow.

Activation and Cultivation Methods

  1. Medium Preparation: Use Potato Dextrose Agar (PDA) medium (purchased from Beina Biology) for cultivation. Add 47.0 g of PDA to 1000 mL of pure water, autoclave at 121°C for 20 minutes, then pour into petri dishes. (Note: Medium components are as follows: potato extract powder: 10.0 g, glucose: 20.0 g, KH₂PO₄: 3.0 g, MgSO₄·7H₂O: 1.5 g, thiamine: 0.008 g, agar: 15.0 g, pH: 6.0±0.2)
  2. Take the test tube containing lyophilized powder from -80°C, place it on ice in a laminar flow hood. Wipe the outer tube with cotton soaked in 75% ethanol, heat the top with an alcohol lamp, then quickly drop sterile water to crack the top. Subsequently, use tweezers to break it open, and remove the inner tube and cotton plug.
  3. Aspirate 0.5 mL of sterile water into the lyophilization tube to fully dissolve the fungal powder.
  4. Transfer the solution to a microcentrifuge tube (EP tube), repeat the process with another 0.5 mL of sterile water, seal the tube, and let it stand at room temperature for 1 hour.
  5. Add 200 μL of the fungal solution to each petri dish and spread it evenly.
  6. Incubate the petri dishes at 25–28°C for 3–5 days; the strain can be used once it grows.

Preparation of Metarhizium Spore Suspension

Metarhizium can produce sufficient spores after culturing on PDA medium for approximately 1 week. Further prepare the spore suspension for subsequent experiments such as transformation, following these steps:

  1. Prepare 0.05% Tween-80, place it in an autoclave, and sterilize at 121°C for 20 minutes.
  2. Take a petri dish with well-grown Metarhizium, add 6 mL of sterilized 0.05% Tween-80 to the dish, gently scrape the surface of the colony with a pipette tip or spreader to detach the spores, and prepare a crude spore suspension.
  3. Take a double-ended plastic tube, cover one end with 2 layers of gauze. Aspirate the crude spore suspension with a pipette, press the pipette tip against the gauze to expel the liquid. Remove hyphae through 2 layers of sterilized gauze to obtain the spore suspension.
  4. Determine the spore concentration using a hemocytometer.
  5. Add an appropriate amount of 0.05% Tween-80 according to the concentration, dilute to the required concentration, and shake well to disperse the spores evenly.

Efficacy Verification of Recombinant Metarhizium Strains

Total RNA Extraction from Fungi

  1. Add 1 mL of Buffer Rlysis-F to a 1.5 mL RNase-free microcentrifuge tube and set aside.
  2. Take 20 mg of dried hyphae, grind them into powder in liquid nitrogen, add the powder to the aforementioned 1.5 mL microcentrifuge tube, and immediately shake to mix thoroughly.
  3. Add 200 μL of chloroform to the lysed sample, mix well. Centrifuge at 12,000 rpm at 4°C for 5 minutes, then collect 600 μL of the supernatant.
  4. Add 200 μL of absolute ethanol, mix well, let stand at room temperature for 3 minutes, centrifuge at 12,000 rpm at 4°C for 5 minutes, and discard the supernatant.
  5. Add 700 μL of 75% ethanol (prepared with DEPC-treated ddH₂O) to the precipitate for washing, centrifuge at 12,000 rpm at 4°C for 3 minutes, and discard the supernatant.
  6. Repeat Step 5 once.
  7. Invert the tube at room temperature for 10 minutes to air-dry the ethanol.
  8. Add 50 μL of DEPC-treated ddH₂O to dissolve the precipitate, obtaining pure RNA.

RT-PCR Verification

Perform the reverse transcription reaction according to the following system and procedure (Table 21)

Table 21

The reverse transcription product was used for PCR verification (Table 22)

Table 22

Detection Module – Bacillus subtilis Construction Experiment

Activation and Cultivation of Bacterial Strains

  1. Prepare LB solid medium without antibiotics. LB agar medium was purchased from Haibo Biology. Each liter contains 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, and 15 g agar. For use, dissolve 35 g of the medium in 1 L pure water, autoclave at 121°C, and then pour into Petri dishes to form plates.
  2. The glycerol stock of Bacillus subtilis strain 168Δ4 was obtained from Biyuntian (Cat. No.: D0441). Remove the glycerol stock and thaw it on ice, then transfer it to a biosafety cabinet. All subsequent operations were performed inside the biosafety cabinet: Immerse a 200 μL sterile pipette tip in 75% ethanol, then flame-sterilize the tip in ethanol to ensure sterility of the tip's end. Next, use the tip to dip a small amount of the glycerol stock, and perform continuous Z-shaped streaking on the LB plate at an angle parallel to the plate. Replace with a new sterile 200 μL pipette tip, and perform continuous Z-shaped streaking along the original streaks. Incubate the plate upside down at 37°C overnight.
  3. Prepare LB liquid medium without antibiotics. LB liquid medium was purchased from Haibo Biology. Each liter contains 10 g tryptone, 5 g yeast extract, and 5 g sodium chloride. For use, dissolve 25 g of the medium in 1 L pure water, and autoclave at 121°C for 15 minutes.
  4. Take the LB solid medium (without antibiotics) containing newly activated single bacterial colonies. Use tweezers to hold a sterile plastic pipette tip, pick one single colony from the plate, and place the tip (with the bacterial colony attached) into a 10 mL test tube containing 3 mL LB liquid medium. Incubate at 37°C with shaking at 200 rpm overnight.

Identification of Transformants

  1. Take the successfully transformed E. coli DH5α that was cultured overnight at 37°C. Use a sterile pipette tip to pick single colonies from the plate obtained in the previous step, and add them to the following PCR system (Table 23). The sequences of the primers are as follows:
    • F-primer (5'-3'): GACCTCGTTTCCACCGGAAT
    • P-primer (5'-3'): TCTTCTTCCGTGATTCCTTG
Table 23

Plasmid Transformation and Screening

Plasmid Transformation into Bacillus subtilis

  1. Remove the prepared Bacillus subtilis competent cells from -80°C, thaw them on ice, then add 5 μg of plasmid to each tube. Mix gently and incubate at 37°C with shaking at 200 rpm for 120 minutes.
  2. Centrifuge at 4,000×g for 1 minute to collect the bacterial pellet, retain 50 μL of supernatant to resuspend the pellet, and spread the suspension onto LB solid medium containing 5 μg/mL chloramphenicol (Cmᵣ). Incubate the plate upside down at 37°C overnight.

Identification of Transformants

  1. On the next day, use a sterile pipette tip to pick single colonies from the plate and inoculate them into LB liquid medium containing 5 μg/mL chloramphenicol (Cmᵣ). Incubate at 37°C with shaking at 200 rpm for 24 hours.
  2. Extract plasmids by following the protocol of the Tiangen Plasmid Extraction Kit (Cat. No.: DP103).
  3. Prepare the PCR system according to the procedure in Section 7.3.2 and perform PCR.
  4. To further improve the reliability of the results, conduct plasmid restriction enzyme digestion verification simultaneously. Prepare the following restriction digestion system (Table 24), then incubate the reaction mixture in a 37°C water bath for 30 minutes.
Table 24

Sucrose Induction and Metabolite Extraction

  1. Inoculate the overnight-cultured Bacillus subtilis transformants into LB liquid medium containing 5 μg/mL chloramphenicol (Cmᵣ) at a 1:100 ratio, and incubate at 37°C with shaking at 200 rpm for 2 hours.
  2. For the recombinant plasmid group, add sucrose to a final concentration of 0%, 4%, 6%, and 8% (w/v), respectively, and incubate at 37°C with shaking at 200 rpm for 24 hours.
  3. After adjusting the OD₆₀₀ of all bacterial cultures to 2.0, centrifuge at 3,000×g for 5 minutes at 4°C, and place the samples on ice immediately.
  4. For every 200 μL of supernatant, add 800 μL of acetonitrile to precipitate proteins and extract methyl salicylate.
  5. Centrifuge at 21,000×g for 3 minutes at 4°C; the resulting supernatant is used for LC-MS analysis.

LC-MS Analysis

  1. A Waters BEH C18 column (1.0 mm × 50 mm, 1.7 μm) was connected to a Dionex Ultimate 3000 system. The column oven temperature was maintained at 25°C throughout the 5-minute isocratic elution (50% mobile phase B) process.
  2. The experiment was conducted at a flow rate of 100 μL/min, using the following mobile phases: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile. The injection volume was set to 1 μL for all analyses.
  3. The output signal of the liquid chromatograph was connected to a Thermo Scientific Q Exactive HF mass spectrometer, which was operated in heated electrospray ionization (HESI) mode. The data acquisition duration was 5 minutes, with parallel reaction monitoring (PRM) ion transitions configured as follows: methyl salicylate was detected in positive ion mode within 1.8–5 minutes. The spray voltage was 3.5 kV, the capillary temperature was set to 320°C, the sheath gas flow rate was 35 (arb. units), the auxiliary gas flow rate was 10 (arb. units), and the maximum spray current was 100 μA. High-frequency sampling of PRM ion transitions was performed at a resolution of 15,000, with an automatic gain control (AGC) target value of 3×10⁶ and a maximum integration time (IT) of 500 ms. The fixed primary mass was 50 m/z, the isolation window width was 0.4 m/z, and the normalized collision energy (nCE) was fixed at 35. All data were acquired in profile mode, and peak height detection was performed using a custom Thermo Scientific Quan Browser method developed based on standard substances.

Materials & Instruments

Materials required for the experiment: plasmid DNA, cell culture medium, transfection reagent, PCR primers, sequencing kit, etc. The main instruments include PCR instrument, centrifuge, flow cytometer, sequencer, etc.

Materials & Instruments

Materials List

Name Company Catalog Number
amino acid substances Shanghai Macklin Biochemical Co., Ltd. A810002-25g
Isopropyl alcohol Macklin I805819-500ml
Coomassie Brilliant Blue G-250 Biosharp BS044-10g
Ethanol anhydrous Macklin E803844-500ml
Chloroform : Isoamyl alcohol (24:1) Macklin C816748-100ml
AG RNAex Pro Reagent Accurate Biotechnology (Hunan) Co., Ltd AG21102
PureLink RNA Mini Kit Invitrogen, Thermo Fisher Scientific 12183018A
TransScript One-Step gDNA and cDNA Synthesis SuperMix TransGen Biotech AT311-02
PerfectStart Green qPCR SuperMix Tiangen Biotech (Beijing) Co., Ltd AQ101-01
Tris-Glycine-SDS Vazyme PS105
TBS/Tween Buffer 10x Vazyme PS103
BL21 (DE3) Escherichia coli cells TIANGEN BIOTECH (BEIJING) CO.,LTD. CB105-02
Chloramphenicol Sangon Biotech A600118
2 × Taq Master Mix (Dye Plus) Vazyme P112-01
IPTG Sangon Biotech A600168
10x Phosphate-buffered Saline (PH 6.8) Biosharp BL551A
Protease Inhibitor Cocktail, Universal, 100× Biosharp BL612A
Lysozyme Beyotime P2226-5
Nuclease-free Water Biosharp BL510A
Benzonase Nuclease Beyotime D7121-25KU
0.22 μm polyethersulfone (PES) syringe filter Biosharp BS-PES25-22-S
His-tag Protein Purification Kit Beyotime P2226
5x SDS-PAGE Sample Loading Buffer Biosharp BL502B
Acetic acid glacial MACKLIN A801295
Bradford Protein Assay Kit Beyotime P0006
RNase A Thermo Scientific™ EN0531
RiboLock RNase Inhibitor Thermo Scientific™ EO0381
Isopropyl Alcohol MACKLIN I766839
EDTA Thermo Scientific™ O2793500
Ethanol [Anhydrous] MACKLIN E809056
DL1000 DNA Marker TAKARA 3591Q
DL 500 DNA Marker Sangon Biotech B600303
Brilliant Blue R MACKLIN B802269
Phosphotungstic acid hydrate Millipore 79690-25G
Dual-color Prestained Low Molecular Weight Protein Ladder (1.2~40 kDa) epizyme WJ402
Unstained Protein Molecular Weight Marker (Pierce™) Thermo Scientific™ 26610
OCT Embedding Matrix Sakura 4583
Ethanol, Absolute Sinopharm Chemical Reagent Co., Ltd. 100092683
Triton® X-100 Permeabilization Solution BioFroxx 1139ML100
Normal Goat Serum Boster Biological Technology Co., Ltd. AR1009
TBS Buffer Salt Powder Bioss Biotech Co., Ltd. BP0152
DAPI Solution Solarbio C0060
Fluorescent Mounting Medium Southern Biotech 0100-01
EGFP Antibody Oasis biofarm OB-PGP003
LB Agar(lennox) Qingdao Hope Bio-Technology Co., Ltd. HB0129-2
LB Broth Qingdao Hope Bio-Technology Co., Ltd. HB0128
Agarose Biosharp, Labgic Technology Co., Ltd. BS081-100g
DNase Ⅰ, RNase-free Thermo Fisher Scientific EN0521
DNase I Beyotime Biotech Inc D7073
RNase T1 Thermo Fisher Scientific EN0541
FastDigest EcoRⅠ Thermo Fisher Scientific FD0274
Goldenview Biosharp, Labgic Technology Co., Ltd. BS357A
Superred Biosharp, Labgic Technology Co., Ltd. BS354B
T7 RNA Transcription Kit Plus belong SJ001V2-01
FastDigest BamHⅠ Thermo Fisher Scientific FD0055
TAE Buffer (50x) Biosharp, Labgic Technology Co., Ltd. BL533A
DNase and RNase AWAY Beyotime Biotech Inc B0123
RNaseEraser Foregene Co., Ltd. RC-01011
Nuclease-free Water Biosharp, Labgic Technology Co., Ltd. BL510B
Plasmid Extraction Kit (Centrifugation Column Type) TIANGEN DP103-03
Ampicillin biosharp BS112
Kanamycin biosharp BS134
FastDigest NheⅠ Thermo Fisher Scientific FD0974
FastDigest ApaⅠ Thermo Fisher Scientific FD1414
FastDigest XbaⅠ Thermo Fisher Scientific FD0684
DL2,000 DNA Marker Takara 3427Q
DL5,000 DNA Marker Takara 3428Q
DNA Loading Buffer (6×) Biosharp, Labgic Technology Co., Ltd. BL532A
PDA Medium Bena Culture Collection 370555
Tween-80 Sangon Biotech A600562
DH5α Competent Cells Weidi Biotechnology P815662
Kanamycin Sulfate Sangon Biotech A506636
Agrobacterium AGL1 Competent Cells Weidi Biotechnology AC1020
Fungal Genomic DNA Extraction Kit Sangon Biotech B518229
KH₂PO₄ Macklin P816382
K₂HPO₄ Macklin M813597
MgSO₄·7H₂O Macklin C804986
CaCl₂·2H₂O Macklin S805275
NaCl Macklin A801012
(NH₄)₂SO₄ Macklin F809847
FeSO₄·7H₂O Macklin A601111
Acetosyringone Sangon Biotech A100169
MES Sangon Biotech B0394
H₃BO₃ sigma A500331
MnCl₂·4H₂O Sangon Biotech 767420
Na₂MoO₄·2H₂O Macklin Z820755
ZnCl₂ Macklin I809489
FeCl₃·6H₂O Macklin C805353
CuSO₄·5H₂O Macklin S818055
Na₂SO₄ Macklin P816348
KCl Macklin C805758
CaCl₂, anhydrous Macklin 221295
KNO₃ sigma A362623
Glufosinate Sangon Biotech F556549
Cefotaxime sodium Sangon Biotech A430167
Peptone Macklin P914715
Dimethyl sulfoxide Macklin D806645
Sterile Filter Membrane Huankai 21624
75% Ethanol Macklin E809056
IM Induction Medium (Liquid) Ararat 58242
IM Induction Medium (Solid) Ararat 69261
Fungal Total RNA Rapid Extraction Kit Sangon Biotech B518629
Bacillus subtilis 168Δ4 Glycerol Stock Beyotime D0441
Methyl Salicylate Shanghai Macklin Biochemical Co., Ltd. M816819
Acetonitrile Macklin A805878
Formic Acid Macklin F805088
Sucrose Macklin S814888
GM Ⅰ Medium, GM Ⅱ Medium Chinook GM-001
LB agar medium Qingdao Hope Bio-Technology Co., Ltd. GM-002
LB broth medium Qingdao Hope Bio-Technology Co., Ltd. HB0128
Chloramphenicol Sangon Biotech HB0129
Ampicillin Sangon Biotech C602545
CaCl₂ Chinook A600199
MgCl₂ Chinook C1001
BamHI Takara M1002
ApaⅠ Takara 1610A
Fast digest buffer Takara 1041A

Instruments

Instrument / Equipment Name Company / Manufacturer
QuantStudio 5 Real-Time PCR System Thermo Fisher Scientific
Biometra TAdvanced (PCR System) Analytik Jena
Precision Balance, 320g Capacity, 1mg Readability, BSA323S-CW Sartorius Thailand Co., Ltd.
Centrifuge 5430 - Microcentrifuge EPPENDORF CO., LTD.
Centrifuge 5418 R (Refrigerated Centrifuge) EPPENDORF CO., LTD.
D1008 Low Speed Centrifuge (Mini Centrifuge) DLAB SCIENTIFIC CO., LTD.
NanoPhotometer N50 (Ultramicrospectrophotometer) Implen GmbH
HYK Dry Bath (Constant Temperature Metal Bath HYK0032) HYK Huayinkang
UVP Transilluminator (Benchtop UV Transilluminator) Analytik Jena GmbH + Co. KG
Electrically Heated Constant Temperature Incubator SHANGHAI BLUEPARD INSTRUMENTS CO., LTD.
Isotherm® Forced Convection Lab Oven Esco
Steam Sterilizer (Laboratory Use, Large Capacity, Vertical Autoclave) YAMATO SCIENTIFIC CO., LTD.
Benchtop Incubator Shaker (Constant Temperature Oscillating Incubator ZQTY-70N) Shanghai Zhichu General Equipment Development Co., Ltd.
Ultrasonic Homogenizer JY92-IIN (Ultrasonic Cell Disruptor) Ningbo Scientz Biotechnology Co., Ltd.
Drill (Drilling Machine) DELIXI GROUP CO., LTD.
Zeiss Axioplan II Fluorescence Microscope Zeiss
Transference Decoloring Shaker (Side-to-Side Shaker) Beyotime
Multiskan FC Microplate Photometer Thermo Scientific™
Mini-PROTEAN® Tetra Cell System (Vertical Electrophoresis Cell) BIO-RAD
Cryostat (Frozen Section Machine) Thermo Fisher Scientific
Anti-Drop Microscope Slides Bioss Biotech Co., Ltd.
Fluorescence Microscope Olympus Corporation
Imaging System 3D HISTECH Ltd.
Pipette Gun (Micropipette) DLAB Scientific Co., Ltd.
Histochemical Pen Fuzhou Maixin Biotech Co., Ltd.
LC System Dionex Ultimate 3000
Column Waters ACQUITY BEH C18, 1.0 mm × 50 mm, 1.7 µm
Mass Spectrometer (MS) Thermo Scientific Q Exactive HF with HESI source

Sequences

The experimental procedure includes the following steps: cell culture, plasmid construction, gene transfection, gene editing, cell screening, sequencing analysis, data processing, and other steps.

Sequence

In this section, we have systematically organized the sequences into the following 5 modules. All sequences mentioned in our project can be found in these 5 modules.

RNA Molecules with Silencing Function

In this module, you can find all RNA molecule sequences with silencing function. You can quickly locate the sequence you need based on the name of the RNA or the name of the target it silences.

Name Application Sequence (5'→3')
dsCHS Single-target dsRNA for silencing the CHS gene in brown citrus aphid UUGAUACCUCUCAAACGUUGUAGGUGUUUGACGAUAACGACUGCUUGUUUGUCUAGUAGACCAUCGUGGGUCAAAUCUUCAGUCUUUUUGUUGCAACACAAGUUUAAGUCCGUCGAAGCAAGUAUGUGAGACAAAGUACCGAAUCGAUGGAACAACAUGGCGGUAAACUGAACUACUAUGAUGAACGCGAAGAAGAACACAAACACCAAACCAAUGGGUUCCAAGUGUAAGUACUCUUUGCUGAUAAUGAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGC
dsCP Single-target dsRNA for silencing the CP19 gene in brown citrus aphid UUAAAAGUCGAACGCGUCGUUGCUGGGCUUCCUGUAUCCUGUGUUUCCGCUAUCGGACCGGCUAACCACCGCGUUGAAACCUGUUUCUGGGUCUGCGGUGUACUCGACCAGCCUCAUGGCGCCAUCGGGUUCCAGAACACUGUACGCUCCCUUGACCACAUCACCCUCUCGCGCUUCCCAUUGGCUCUUCACGUCGUAAGUUUCCGGAUCGUGAACCGCGUAAUCGAACUUGUACGCUUUGGGUGCGUAAUUUUCUGCAGACUCUGAGCUUUGAGAUCGGGGUCCUCCUUCAACGAAAAUCGGUAUGGCCAUGGUGCAUGCCGUCAUUACGCUCAGCGCUGCUAAAAUUUGACACGACCUCAU
dsCYP450 Single-target dsRNA for silencing the CYP450 gene in brown citrus aphid AACAAAGUGUCAACAGUCAUUCCAAUUACGUCUUUGUAAUCUGCCUCGUCUGAGCUCAAGUAUUCUCCUAAUAAUGAUGCUUUGGAGUCAGUCGUUUUAAUACUGGUUAGUUUUUCACUUACAACUUUUAGGGCCAUUUUUUCAAUGUAUUCGUGUCCUUUUUUUAUACUUUUAUACAUGGGGGUAUCAAAUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUUCAUCGAAGGC
dsF2 Dual-target fusion dsRNA for silencing CHS and CYP450 genes in brown citrus aphid AGCUAACAAAGUGUCAACAGUCAUUCCAAUUACGUCUUUGUAAUCUGCCUCGUCUGAGCUCUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGCUCAUCGAUCGGAUAUAAAUAUUUGUCUAUCAGAUCUCGCCAGAACUGUUGUUUCGGAGUAUCGACUUUAACAUUUCCAUCAUUUCUUCGUGGGUUUCGUGCCACAUGGUCGCCACAGAUAUUGUUUCAUAAUAUUCGUCUGGUUCUUUAUCUCGAUCGGCCAA
dsF3 Triple-target fusion dsRNA for synergistic silencing of CHS, CYP450 and CP19 genes in brown citrus aphid GUUGCUGGGCUUCCUGUAUCCUGUGUUUCCGCUAUCGGACCGGCUAACCAUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGCUCAUCGAUCGGAUAUAAAUAUUUGUCUAUCAGAUCUCGCCAGAACUGUUGUUUCGGAGUAUCGACUUUAACAUUUCCAUCAUUUCUUCGUGGGUUUCGUGCCACAUGGUCGCCACAGAUAUUGUUUCAUAAUAUUCGUCUGGUUCUUUAUCUCGAUCGGCCAA
tri-shRNA Triple tandem shRNA for combined silencing of CHS, CYP450 and CP19 genes in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) ACAUGGGUAACCUCAUGU UCUAGA CGCCGUCGAACAAUUUACAACUUAG UUCAAGAGA CUAAGUUGUAAAUUGUUCGACGGCG AGGCAU GAGCUCAGACGAGGCAGAUUACAAA UUCAAGAGA UUUGUAAUCUGCCUCGUCUGAGCUC GAAUUC GAGCGUACAGUGUUCUGGAACCCGA UUCAAGAGA UCGGGUUCCAGAACACUGUACGCUC
bi-amiRNA Double tandem amiRNA for combined silencing of CHS and CYP450 genes in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) ACAUGAGGUUACCCAUGU AGUUCU CUCGAGUCUGCUCCGUCUAAUGUUU CGGUAUAUUUAAGAU AAACAUUAGACGGAGGAGACUCGAG UG UCCGUA UUCAACAUUUAUCAAGCUGCGGC GCUGGCGCCUGC GCGGCAGCUUGUUAAAUGUUGAAUC
amiRNA amiRNA for silencing the CHS gene in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) gu cgccgucgaucaauuuacaacuuag uagaauuuauauggc cuaaguuguaaauuguucgacggcg ucuuga acauggguaauccucauguuu

Plasmid Sequences

In this module, you can find the sequences of plasmids constructed throughout the entire project. You can quickly locate the sequence you are looking for based on the plasmid's name or its function.

Plasmid Name Function Complete Vector Sequence (5'→3')
pUC57_pac-bi-amiRNA DNA sequence for producing bi-amiRNA molecules (bold sequence is pac site, underlined sequence is target) tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagctaatacgactcactataggccacagcatacatgggtaacctcatgttcaagagagctcagacgaggcagattacaaagccatataaattctatttgtaatctgcctcctctgagctcacaggcataagttgtaaatagttcgacgccgcgaccgcggacgcgccgtcgaacaatttacaacttagggaagagcggatcctctagagggcccgtttaaacccgctgatcagcctcgactctagcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagctcagatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaagcccaatctgaataatgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgaggcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtttttccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca
pUC57_mini-Kana-BsmBI⁻-terminator-ΔT7 Serves as the basic original cloning backbone, with subsequent insertion of corresponding target fragments tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactCTAGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTCAGatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaagcccaatctgaataatgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgaggcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtttttccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca
pUC57_pac-tri-shRNA DNA sequence for producing tri-shRNA (CHS+CYP450+CP) (bold sequence is pac site, underlined sequence is target) tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagctaatacgactcactataggagcatacatgggtaacctcatgttctagacgccgtcgaacaatttacaacttagttcaagagactaagttgtaaattgttcgacggcgaggcatgagctcagacgaggcagattacaaattcaagagatttgtaatctgcctcgtctgagctcgaattcgagcgtacagtgttctggaacccgattcaagagatcgggttccagaacactgtacgctcccacggaagagcggatcctctagagggcccgtttaaacccgctgatcagcctcgactctagcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagctcagatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcaggatcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca
pACYCDuet_1-CP-Histag-CP Can express His-tagged MS2 capsid protein dimer fusion protein GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGTACCCATCACCATCACCATCACGGTACCGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCAgcGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATAGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA
pET28a_CP-GBP3.1-CP-TAT-Histag Can express His-tagged single-chain capsid protein dimer fused with GBP3.1 (aphid intestinal targeting peptide) and TAT (cell-penetrating peptide) TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGATCCGCTAGTAACTTCACCCAATTTGTTCTGGTTGATAATGGCGGCACCGGTGACGTAACCGTGGCTCCGTCTAACTTCGCGAACGGCGTCGCGGAATGGATTTCCAGCAACAGCCGCTCGCAAGCGTATAAGGTGACGTGCAGCGTTCGTCAGAGCTCGGCTCAGAACCGCAAGTACACGATCAAGGTCGAGGTTCCGAAAGTGGCGACGCAAACCGTTGGTGGCGTGGAGCTGCCGGTCGCTGCTTGGCGCAGCTATCTGAACATGGAATTGACCATCCCGATCTTTGCGACCAATTCCGACTGTGAACTGATTGTGAAAGCAATGCAGGGCTTGCTGAAGGACGGCAACCCGATCCCGAGCGCGATCGCAGCGAATTCTGGTATTTACACCTGCAGCAAAAAATACCCGCGTTCCCCGTGTATGGGTGACGTGACTGTAGCGCCAAGCAACTTTGCCAACGGTGTCGCTGAATGGATCAGCTCTAACAGTCGCTCCCAGGCATACAAGGTGACTTGCTCCGTGCGTCAAAGCAGCGCCCAAAACCGTAAATATACCATTAAGGTGGAGGTGCCGAAGGTTGCGACCCAGACCGTTGGTGGCGTTGAGCTCCCTGTTGCCGCGTGGCGTAGCTACCTGAATATGGAATTGACCATTCCGATCTTCGCGACGAACAGCGATTGCGAGTTAATTGTTAAAGCCATGCAGGGTCTGCTGAAAGATGGTAATCCGATCCCATCTGCCATCGCCGCAAATTCCGGCATTTACCAGCTGCAGGGCGGTCCGGCACCGGGTGGTAGCGCGTATGGTCGTAAGAAACGTCGCCAACGTCGTAGACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
pACYCDuet_1-CP-Histag-CP-shRNA Can express intact His-tagged virus-like particles (VLPs) that encapsulate shRNA with a pac site ggggaattgtgagcggataacaattcccctgtagaaataattttgtttaactttaataaggagatataccatggcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaacggggtcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaagtggcaacccagactgttggtggtgtagagcttcctgtagccgcatggcgttcgtacttaaatatggaactaaccattccaattttcgctacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacgctaactttactcagttcgttctcgtcgacaatggcggtacccatcaccatcaccatcacggtaccggcgacgtgactgtcgccccaagcaacttcgctaacggggtcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaagtggcaacccagactgttggtggtgtagagcttcctgtagccgcatggcgttcgtacttaaatatggaactaaccattccaattttcgctacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccagcggccgcataatgcttaagtcgaacagaaagtaatcgtattgtacacggccgcataatcgaaattaatacgactcactataggggcagcggcagctagttaaatgttgaatcatcttaaatataccggattcaacatttaacaagctgccgcagaacttgtacccattaggagtacaaacctaggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaacctcaggcatttgagaagcacacggtcacactgcttccggtagtcaataaaccggtaaaccagcaatagacataagcggctatttaacgaccctgccctgaaccgacgaccgggtcgaatttgctttcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacagacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatagtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacggaactccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcggcgcaaagtgcgtcgggtgatgctgccaacttactgatttagtgtatgatggtgtttttgaggtgctccagtggcttctgtttctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacggcaaaagcaccgccggacatcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatataagttgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaaattaatacgactcactata
pET28a_GBP3.1-EGFP-TAT-Histag Can express His-tagged enhanced green fluorescent protein (EGFP) fused with GBP3.1 (aphid intestinal targeting peptide) and TAT (cell-penetrating peptide) TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCACATGTTCAAAAAAGTATCCCAGGTCTCCGTGTATGGGTGGCGGTGGATCTGGTGGTGGTGGCTCAGGTGGTGGCGGCTCCGGTGGTGGTGGCAGCATGGTGTCTTTTAAGAGCCTGTTGGTGCTGTGTTGCGCGGCGCTGGGCGCATTCGCGACCAAACGTATGAGCAAAGGCGAGGAATTGTTTACGGGTGTCGTGCCAATTCTGGTGGAACTGGATGGCGACGTTAACGGCCACAAGTTCTCCGTTAGCGGTGAGGGCGAGGGCGACGCCACCTACGGTAAATTGACCTTAAAATTCATCTGCACCACCGGTAAGCTGCCGGTTCCGTGGCCTACATTGGTAACGACGTTTACGTACGGCGTTCAATGCTTTAGCCGTTATCCGGATCACATGAAACGCCACGATTTTTTCAAGTCTGCTATGCCGGAAGGCTATGTTCAAGAGCGCACCATTTTCTTTAAGGACGACGGCAACTACAAAACCCGTGCGGAAGTGAAATTCGAGGGTGACACCCTGGTGAATCGTATTGAACTCAAGGGTATTGATTTCAAGGAGGACGGTAACATTCTGGGTCATAAGCTGGAGTATAACTACAACAGCCATAATGTCTATATCATGGCAGATAAGCAGAAAAACGGTATCAAGGCTAATTTTAAGACCAGACACAACATCGAAGATGGCGGAGTCCAGCTGGCGGACCACTACCAGCAGAACACTCCGATCGGCGACGGCCCAGTTCTGTTACCGGATAATCATTACCTTAGCACCCAAAGCGCGCTGAGCAAGGACCCGAATGAAAAACGTGATCACATGGTTCTGTTGGAGTTCGTGACCGCTGCCGGTATCACCCACGGTATGGACGAATTGTATAAACTGCAAGGTGGTCCGGCACCGGGTGGCTCGGCGTACGGCCGTAAAAAACGCCGTCAGCGTCGCCGGCTGCAAGGTGGTCCGGCACCGGGTGGCTCGGCGCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
pET28a_EGFP-Histag Can express His-tagged enhanced green fluorescent protein (EGFP) TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCCTCCAGGGTGGTCCGGCACCGGGTGGCAGCGCTATGGTGAGCTTTAAGTCGCTGCTGGTCTTATGCTGCGCCGCGCTGGGCGCATTCGCGACCAAACGTATGAGCAAGGGTGAGGAACTGTTTACCGGCGTGGTTCCGATCCTGGTTGAATTAGATGGCGACGTGAACGGTCACAAGTTCTCTGTTTCTGGTGAGGGTGAAGGAGACGCCACCTACGGGAAATTGACCCTTAAGTTCATCTGCACCACCGGTAAATTGCCGGTTCCGTGGCCTACCTTGGTAACAACGTTTACGTACGGTGTTCAATGTTTTAGCCGTTATCCGGACCACATGAAACGCCACGATTTCTTCAAGTCCGCTATGCCGGAAGGCTATGTGCAAGAGCGCACCATTTTTTTCAAGGACGACGGCAACTACAAGACTCGCGCGGAAGTTAAATTCGAGGGTGACACCCTGGTGAATCGTATTGAGCTGAAAGGTATTGATTTTAAAGAAGATGGAAATATTCTGGGTCATAAGCTGGAGTACAACTATAACAGCCATAATGTTTATATTATGGCAGATAAACAGAAAAACGGCATCAAAGCGAACTTTAAGACGCGTCATAACATCGAGGACGGCGGTGTCCAGCTGGCGGATCACTACCAGCAAAACACCCCGATCGGCGACGGCCCAGTGTTGCTGCCGGATAATCATTACCTGTCCACCCAGAGCGCTCTGTCCAAGGACCCGAATGAAAAACGTGATCACATGGTTCTGCTTGAGTTCGTGACTGCCGCGGGTATCACCCACGGCATGGATGAACTGTATAAGCTCCAGGGTGGTCCGGCACCGGGTGGCAGCGCTCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
pUC57_CHS For producing double-stranded RNA (dsRNA) of the CHS gene (The underlined sequence is the target sequence) TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATCATTATCAGCAAAGAGTACTTACACTTGGAACCCATTGGTTTGGTGTTTGTGTTCTTCTTCGCGTTCATCATAGTAGTTCAGTTTACCGCCATGTTGTTCCATCGATTCGGTACTTTGTCTCACATACTTGCTTCGACGGACTTAAACTTGTGTTGCAACAAAAAGACTGAAGATTTGACCCACGATGGTCTACTAGACAAACAAGCAGTCGTTATCGTCAAACACCTACAACGTTTGAGAGGTATCAACCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
pUC57_CHS-tri-terminator For producing double-stranded RNA (dsRNA) of the CHS gene (The underlined sequence is the target sequence) TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGGTCGACCAGCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGAAGCTTCTTTCAGCAAAAAACCCCGCGAGACCCCCGAAGAGGCCCCGCGGGGTTATGCTAGTAATACGACTCACTATAGGGGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATCATTATCAGCAAAGAGTACTTACACTTGGAACCCATTGGTTTGGTGTTTGTGTTCTTCTTCGCGTTCATCATAGTAGTTCAGTTTACCGCCATGTTGTTCCATCGATTCGGTACTTTGTCTCACATACTTGCTTCGACGGACTTAAACTTGTGTTGCAACAAAAAGACTGAAGATTTGACCCACGATGGTCTACTAGACAAACAAGCAGTCGTTATCGTCAAACACCTACAACGTTTGAGAGGTATCAACCCTATAGTGAGTCGTATTACTAGCATAACCCCGCGGGGCCTCTTCGGGGGTCTCGCGGGGTTTTTTGCTGAAAGAAGCTTCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGCTGGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
pUC57_dsF2 For producing double-stranded RNA (dsRNA) of the dual fusion gene (The underlined sequence is the target sequence) TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTTGGCCGATCGAGATAAAGAACCAGACGAATATTATGAAACAATATCTGTGGCGACCATGTGGCACGAAACCCACGAAGAAATGATGGAAATGTTAAAGTCGATACTCCGAAACAACAGTTCTGGCGAGATCTGATAGACAAATATTTATATCCGATCGATGAGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATACGACTACAGTGGTTCAAAGACGAAGAATGGGACTCTAATTCGGAAAGTTCGAAATTGATCAAAGCAGCTCACGATATAAACAGTGCGATACTGAAGACTGATAATGGACCACAACTATGGAGAAAAGAGCTCAGACGAGGCAGATTACAAAGACGTAATTGGAATGACTGTTGACACTTTGTTAGCTCCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
pUC57_dsF3 For producing dsF3 molecules (The underlined sequence is the target sequence) TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTTGGCCGATCGAGATAAAGAACCAGACGAATATTATGAAACAATATCTGTGGCGACCATGTGGCACGAAACCCACGAAGAAATGATGGAAATGTTAAAGTCGATACTCCGAAACAACAGTTCTGGCGAGATCTGATAGACAAATATTTATATCCGATCGATGAGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATACGACTACAGTGGTTCAAAGACGAAGAATGGGACTCTAATTCGGAAAGTTCGAAATTGATCAAAGCAGCTCACGATATAAACAGTGCGATACTGAAGACTGATAATGGACCACAACTATGGAGAAAATGGTTAGCCGGTCCGATAGCGGAAACACAGGATACAGGAAGCCCAGCAACCCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
pBar_pac-dsF3 For modifying Metarhizium anisopliae to produce dsF3 molecules (The bold sequence is the pac site, and the underlined sequence is the target sequence) cccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcaggatcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatctggatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattgctctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacatgattacgaattccactagtgtcgacagaagatgacattgaaggagcactttttgggcttggctggagctagtggaggtcaacaatgaatgcctattttggtttagtcgtccaggcggtgagcacaaaatttgtgtcgtttgacaagatggttcatttaggcaactggtcagatcagccccacttgtagcagtagcggcggcgctcgaagtgtgactcttattagcagacaggaacgaggacattattatcatctgctgctggtgcacgataacttggtgcgtttgtcaagcaaggtaagtgaacgacccggtcataccttcttaagttcgcccttcctccctttatttcagattcaatctgacttacctattctacccaagcatcgataagcggccgctctagtggatctaccatgagcccagaacgacgcccggccgacatccgccgtgccaccgaggcggacatgccggcggtctgcaccatcgtcaaccactacatcgagacaagcacggtcaacttccgtaccgagccgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggcatcgcctacgcgggcccctggaaggcacgcaacgcctacgactggacggccgagtcgaccgtgtacgtctccccccgccaccagcggacgggactgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgagcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttctggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtcaccgaaatctgaggatccacttaacgttactgaaatcatcaaacagtttgacgaatctggatataagatcgttggtgtcgatgtcagctccggagttgagacaaatggtgttcaggatctcgataagatacgttcatttgtccaagcagcaaagagtgccttctagtgatttaatagctccatgtcaacaagaataaaacgcgtttcgggtttacctcttccagatacagctcatctgcaatgcattaatgcattggacctcgcaaccctagtacgcccttcaggctccggcgaagcagaagaatagcttagcagagtctattttcattttcgggagacgagatcaagcagatcaacggtcgtcaagagacctacgagactgaggaatccgctcttggctccacgcgactatatatttgtctctaattgtactttgacatgctcctcttctttactctgatagcttgactatgaaaattccgtcaccagcccctgggttcgcaaagataattgcactgtttcttccttgaactctcaagcctacaggacacacattcatcgtaggtataaacctcgaaaatcattcctactaagatgggtatacaatagtaaccatgcatggttgcctagtgaatgctccgtaacacccaatacgccggccgaaacttttttacaactctcctatgagtcgtttacccagaatgcacaggtacacttgtttagaggtaatccttctttctagagtcgacctgcagactagtgtcgacagaagatgacattgaaggagcactttttgggcttggctggagctagtggaggtcaacaatgaatgcctattttggtttagtcgtccaggcggtgagcacaaaatttgtgtcgtttgacaagatggttcatttaggcaactggtcagatcagccccacttgtagcagtagcggcggcgctcgaagtgtgactcttattagcagacaggaacgaggacattattatcatctgctgcttggtgcacgataacttggtgcgtttgtcaagcaaggtaagtgaacgacccggtcataccttcttaagttcgcccttcctccctttatttcagattcaatctgacttacctattctacccaagcatcgataattggcgcgccacatgggtaacctcatgtttggccgatcgagataaagaaccagacgaatattatgaaacaatatctgtggcgaccatgtggcacgaaacccacgaagaaatgatggaaatgttaaagtcgatactccgaaacaacagttctggcgagatctgatagacaaatatttatatccgatcgatgagccgataggcgtcaagacaaatattacgactattgcagagacgtctgagatacgactacagtggttcaaagacgaagaatgggactctaattcggaaagttcgaaattgatcaaagcagctcacgatataaacagtgcgatactgaagactgataatggaccacaactatggagaaaatggttagccggtccgatagcggaaacacaggatacaggaagcccagcaacttcaagagacaacgacccgaaggacataggacacaaaggcgatagcctggccgattggtaaaagaggtatcaacaccaggtaatagtcagaagtcatagcgtgacaaatatagcactcgacgaaactagttaaagcttgaaaggcttaatctcagggtaagaagcagaaacttggtgacatcagcatagagtctgcagagacgttatcagcattataaacagaactgcggatagccgagtagctagcctatatttataaacagatagtctagagcggtcttgacaacaaagcctcatagctgaaattgtaaaggtagtaaagaagcacccaaagcacggtgtaccagcggtgtctataacaaagtattataagcagaccaagaaatagagctagccggttctcgagacttaacgttactgaaatcatcaaacagtttgacgaatctggatataagatcgttggtgtcgatgtcagctccggagttgagacaaatggtgttcaggatctcgataagatacgttcatttgtccaagcagcaaagagtgccttctagtgatttaatagctccatgtcaacaagaataaaacgcgtttcgggtttacctcttccagatacagctcatctgcaatgcattaatgcattggacctcgcaaccctagtacgcccttcaggctccggcgaagcagaagaatagcttagcagagtctattttcattttcgggagacgagatcaagcagatcaacggtcgtcaagagacctacgagactgaggaatccgctcttggctccacgcgactatatatttgtctctaattgtactttgacatgctcctcttctttactctgatagcttgactatgaaaattccgtcaccagcccctgggttcgcaaagataattgcactgtttcttccttgaactctcaagcctacaggacacacattcatcgtaggtataaacctcgaaaatcattcctactaagatgggtatacaatagtaaccatgcatggttgcctagtgaatgctccgtaacacccaatacgccggccgaaacttttttacaactctcctatgagtcgtttacccagaatgcacaggtacacttgtttagaggtaatccttcttaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgagcttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttaaagtactttaaagtactttaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgagcggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaacttattaaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccc
pgrac100 Bacillus subtilis-Escherichia coli shuttle plasmid, serving as the original plasmid backbone ttaagttattggtatgactggttttaagcgcaaaaaaagttgctttttcgtacctattaatgtatcgttttagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcaggccttaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgttttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaattagcttggtaccaAAGGAGGTAAGGATCACTAGAAAATTTTTTAAAAAATCTCTTGACATTGGAAGGGAGATATGTTATTATAAGAATTGCGGAATTGTGAGCGGATAACAATTCCCATATAAAGGAGGAAGGATCAATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAGCATCAGCTGCTGGATCCATGTCTAGACTGCAGACTAGTGTCGACGTCGCGGCCGCACATCACCATCACCATCACCATCACTAACGTCCCCggggcagcccgcctaatgagcgggcttttttcacgtcacgcgtccatggagatctttgtctgcaactgaaaagtttataccttacctggaacaaatggttgaaacatacgaggctaatatcggcttattaggaatagtccctgtactaataaaatcaggtggatcagttgatcagtatattttggacgaagctcggaaagaatttggagatgacttgcttaattccacaattaaattaagggaaagaataaagcgatttgatgttcaaggaatcacggaagaagatactcatgataaagaagctctaaaactattcaataaccttacaatggaattgatcgaaagggtggaaggttaatggtacgaaaattaggggatctacctagaaagccacaaggcgataggtcaagcttaaagaacccttacatggatcttacagattctgaaagtaaagaaacaacagaggttaaacaaacagaaccaaaaagaaaaaaagcattgttgaaaacaatgaaagttgatgtttcaatccataataagattaaatcgctgcacgaaattctggcagcatccgaagggaattcatattacttagaggatactattgagagagctattgataagatggttgagacattacctgagagccaaaaaactttttatgaatatgaattaaaaaaaagaaccaacaaaggctgagacagactccaaacgagtctgtttttttaaaaaaaatattaggagcattgaatatatattagagaattaagaaagacatgggaataaaaatattttaaatccagtaaaaatatgataagattatttcagaatatgaagaactctgtttgtttttgatgaaaaaacaaacaaaaaaaatccacctaacggaatctcaatttaactaacagcggccaaactgagaagttaaatttgagaaggggaaaaggcggatttatacttgtatttaactatctccattttaacattttattaaaccccatacaagtgaaaatcctcttttacactgttcctttaggtgatcgcggagggacattatgagtgaagtaaacctaaaaggaaatacagatgaattagtgtattatcgacagcaaaccactggaaataaaatcgccaggaagagaatcaaaaaagggaaagaagaagtttattatgttgctgaaacggaagagaagatatggacagaagagcaaataaaaaacttttctttagacaaatttggtacgcatataccttacatagaaggtcattatacaatcttaaataattacttctttgatttttggggctattttttaggtgctgaaggaattgcgctctatgctcacctaactcgttatgcatacggcagcaaagacttttgctttcctagtctacaaacaatcgctaaaaaaatggacaagactcctgttacagttagaggctacttgaaactgcttgaaaggtacggttttatttggaaggtaaacgtccgtaataaaaccaaggataacacagaggaatccccgatttttaagattagacgtaaggttcctttgctttcagaagaacttttaaatggaaaccctaatattgaaattccagatgacgaggaagcacatgtaaagaaggctttaaaaaaggaaaaagagggtcttccaaaggttttgaaaaaagagcacgatgaatttgttaaaaaaatgatggatgagtcagaaacaattaatattccagaggccttacaatatgacacaatgtatgaagatatactcagtaaaggagaaattcgaaaagaaatcaaaaaacaaatacctaatcctacaacatcttttgagagtatatcaatgacaactgaagaggaaaaagtcgacagtactttaaaaagcgaaatgcaaaatcgtgtctctaagccttcttttgatacctggtttaaaaacactaagatcaaaattgaaaataaaaattgtttattacttgtaccgagtgaatttgcatttgaatggattaagaaaagatatttagaaacaattaaaacagtccttgaagaagctggatatgttttcgaaaaaatcgaactaagaaaagtgcaataaactgctgaagtatttcagcagttttttttatttagaaatagtgaaaaaaatataatcagggaggtatcaatatttaatgagtactgatttaaatttatttagactggaattaataattaacacgtagactaattaaaatttaatgagggataaagaggatacaaaaatattaatttcaatccctattaaattttaacaagggggggattaaaatttaattagaggtttatccacaagaaaagaccctaataaaatttttactagggttataacactgattaatttcttaatgggggagggattaaaatttaatgacaaagaaaacaatcttttaagaaaagcttttaaaagataataataaaaagagctttgcgattaagcaaaactctttactttttcattgacattatcaaattcatcgatttcaaattgttgttgtatcataaagttaattctgttttgcacaaccttttcaggaatataaaacacatctgaggcttgttttataaactcagggtcgctaaagtcaatgtaacgtagcatatgatatggtatagcttccacccaagttagcctttctgcttcttctgaatgtttttcatatacttccatgggtatctctaaatgattttcctcatgtagcaaggtatgagcaaaaagtttatggaattgatagttcctctctttttcttcaacttttttatctaaaacaaacactttaacatctgagtcaatgtaagcataagatgtttttccagtcataatttcaatcccaaatcttttagacagaaattctggacgtaaatcttttggtgaaagaatttttttatgtagcaatatatccgatacagcaccttctaaaagcgttggtgaatagggcattttacctatctcctctcattttgtggaataaaaatagtcatattcgtccatctacctatcctattatcgaacagttgaactttttaatcaaggatcagtcctttttttcattattcttaaactgtgctcttaactttaacaactcgatttgtttttccagatctcgagggtaactagcctcgccgatcccgcaagaggcccggcagtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcatgc
pgrac100-MS Bacillus subtilis-Escherichia coli shuttle plasmid with inserted SacB Promoter, RBS, pchB, pchA, BSMT1, and terminator; converts sucrose to methyl salicylate ttaagttattggtatgactggttttaagcgcaaaaaaagttgctttttcgtacctattaatgtatcgttttagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcaggccttaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgttttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaattagcttggtaccCCGCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGAAAGGAGGAAGGATCCATGATGAAAACACCGGAAGATTGCACAGGCCTGGCAGATATTAGAGAGGCGATTGATCGCATTGATCTGGATATTGTTCAGGCACTGGGCAGAAGAATGGATTATGTTAAAGCAGCATCACGCTTTAAAGCATCAGAAGCAGCAATTCCGGCACCGGAAAGAGTTGCAGCAATGCTGCCGGAAAGAGCAAGATGGGCAGAAGAAAATGGCCTGGATGCACCGTTTGTTGAAGGCCTGTTTGCACAGATCATCCACTGGTATATTGCAGAACAAATTAAATACTGGAGACAGACAAGAGGCGCAGCATGAGCAGACTGGCACCGCTGTCACAATGCCTGCATGCACTGAGAGGCACATTTGAAAGAGCAATTGGCCAAGCACAAGCACTGGATAGACCGGTTCTGGTTGCAGCAAGCTTCGAAATTGATCCGCTTGATCCGCTGCAAGTTTTTGGCGCATGGGATGATAGACAAACACCGTGCCTGTATTGGGAACAACCGGAACTGGCATTCTTTGCATGGGGCTGCGCACTGGAACTGCAAGGCCATGGCGAGCAACGCTTTGCACGTATTGAGGAAAATTGGCAACTGCTGTGCGCAGATGCAGTTGTTGAAGGCCCGCTGGCACCGAGACTGTGCGGAGGATTTAGATTTGATCCGAGAGGCCCGAGAGAAGAACATTGGCAAGCATTTGCAGATGCAAGCCTGATGCTGGCAGGCATTACAGTTCTGAGAGAAGGCGAAAGATATAGAGTTCTGTGCCAACATCTGGCAAAACCGGGCGAAGACGCACTGGCACTGGCAGCATATCATTGCAGCGCACTGCTGAGACTGAGACAACCGGCAAGAAGAAGACCGTCAGGCCCGACAGCAGGCGCACAAGGAGATGCATCAGCACAAGAAAGAAGACAATGGGAAGCAAAAGTTTCAGATGCAGTTTCATCAGTTAGACAAGGCAGATTTGGCAAAGTTGTTCTGGCAAGAACACAAGCAAGACCGCTGGGCGATATTGAACCGTGGCAAGTTATTGAACATCTGAGACTGCAACATGCAGATGCACAACTGTTTGCATGCAGAAGAGGCAATGCATGCTTTCTGGGCGCATCACCGGAAAGACTGGTTAGAATTAGAGCAGGCGAAGCACTGACACATGCACTGGCAGGCACAATTGCAAGAGGCGGCGATGCACAAGAAGATGCAAGACTGGGCCAAGCACTGCTGGATAGCGCAAAAGATAGACATGAACATCAACTGGTTGTTGAAGCAATTCGCACAGCACTGGAACCGTTTTCAGAAGTTCTGGAAATTCCGGATGCACCGGGCCTGAAAAGACTGGCAAGAGTTCAACATCTGAATACACCGATTAGAGCAAGACTGGCAGATGCAGGCGGCATTCTGAGACTGCTGCAAGCACTGCATCCGACACCGGCAGTTGGCGGCTATCCGAGATCAGCAGCACTGGATTATATTAGACAACATGAAGGCATGGATAGAGGCTGGTATGCAGCACCGCTGGGCTGGCTGGATGGCGAAGGAAATGGCGATTTTCTGGTTGCACTGAGATCAGCACTGCTGACACCGGGCAGAGGCTATCTGTTTGCAGGCTGCGGCCTGGTTGGAGATTCAGAACCGGCACATGAATATCGCGAAACATGCCTGAAACTGTCAGCAATGAGAGAAGCACTGTCAGCAATTGGCGGCCTGGATGAAGTTCCGCTGCAAAGAGGCGTTGCATAATCTAGAATGGAAGTTGTTGAAGTTCTGCATATGAATGGCGGCAATGGCGATTCAAGCTATGCAAATAACTCACTGGTTCAACAGAAGGTGATTCTGATGACAAAACCGATTACAGAACAAGCAATGATTGACCTGTATTCAAGCCTGTTTCCGGAAACACTGTGCATTGCAGATCTGGGCTGCTCACTGGGCGCAAATACATTTCTGGTTGTTTCACAACTGGTTAAAATCGTTGAGAAGGAGCGCAAAAAGCATGGCTTTAAAAGCCCGGAATTCTATTTCCATTTTAACGACCTGCCGGGCAATGATTTTAACACGCTGTTTCAATCACTGGGCGCATTTCAAGAGGACCTGAGAAAACATATTGGCGAATCATTTGGCCCGTGCTTTTTCTCAGGCGTTCCGGGCTCATTTTATACACGCCTGTTTCCGTCAAAGAGCCTGCATTTTGTTTATTCAAGCTATAGCCTGATGTGGCTGTCACAAGTGCCGAATGGCATTGAAAATAACAAAGGCAATATCTATATGGCACGCACAAGCCCGCTGAGCGTTATTAAAGCATACTACAAGCAGTACGAAATTGACTTCAGCAACTTCCTGAAGTACCGCTCAGAAGAACTGATGAAAGGCGGCAAAATGGTTCTGACACTGCTGGGCAGAGAATCAGAAGATCCGACAAGCAAAGAATGCTGCTATATTTGGGAACTGCTGGCAATGGCACTGAATAAGCTGGTTGAAGAGGGCCTGATTAAAGAAGAAAAAGTTGATGCATTTAACATCCCGCAGTATACGCCGAGCCCGGCAGAGGTTAAGTATATCGTCGAAAAGGAGGGCTCATTTACAATTAATCGCCTGGAAACATCAAGAGTTCATTGGAACGCATCAAACAATGAAAAGAACGGCGGCTATAATGTTAGCAGATGCATGAGAGCAGTTGCAGAACCGCTGCTGGTTTCACATTTTGATAAAGAACTGATGGATCTGGTCTTCCACAAGTACGAAGAGATTGTTTCAGATTGCATGAGCAAAGAAAACACGGAGTTCATTAACGTGATCATCTCACTGACGAAAATCAATTGACCCggggcagcccgcctaatgagcgggcttttttcacgtcacgcgtccatggagatctttgtctgcaactgaaaagtttataccttacctggaacaaatggttgaaacatacgaggctaatatcggcttattaggaatagtccctgtactaataaaatcaggtggatcagttgatcagtatattttggacgaagctcggaaagaatttggagatgacttgcttaattccacaattaaattaagggaaagaataaagcgatttgatgttcaaggaatcacggaagaagatactcatgataaagaagctctaaaactattcaataaccttacaatggaattgatcgaaagggtggaaggttaatggtacgaaaattaggggatctacctagaaagccacaaggcgataggtcaagcttaaagaacccttacatggatcttacagattctgaaagtaaagaaacaacagaggttaaacaaacagaaccaaaaagaaaaaaagcattgttgaaaacaatgaaagttgatgtttcaatccataataagattaaatcgctgcacgaaattctggcagcatccgaagggaattcatattacttagaggatactattgagagagctattgataagatggttgagacattacctgagagccaaaaaactttttatgaatatgaattaaaaaaaagaaccaacaaaggctgagacagactccaaacgagtctgtttttttaaaaaaaatattaggagcattgaatatatattagagaattaagaaagacatgggaataaaaatattttaaatccagtaaaaatatgataagattatttcagaatatgaagaactctgtttgtttttgatgaaaaaacaaacaaaaaaaatccacctaacggaatctcaatttaactaacagcggccaaactgagaagttaaatttgagaaggggaaaaggcggatttatacttgtatttaactatctccattttaacattttattaaaccccatacaagtgaaaatcctcttttacactgttcctttaggtgatcgcggagggacattatgagtgaagtaaacctaaaaggaaatacagatgaattagtgtattatcgacagcaaaccactggaaataaaatcgccaggaagagaatcaaaaaagggaaagaagaagtttattatgttgctgaaacggaagagaagatatggacagaagagcaaataaaaaacttttctttagacaaatttggtacgcatataccttacatagaaggtcattatacaatcttaaataattacttctttgatttttggggctattttttaggtgctgaaggaattgcgctctatgctcacctaactcgttatgcatacggcagcaaagacttttgctttcctagtctacaaacaatcgctaaaaaaatggacaagactcctgttacagttagaggctacttgaaactgcttgaaaggtacggttttatttggaaggtaaacgtccgtaataaaaccaaggataacacagaggaatccccgatttttaagattagacgtaaggttcctttgctttcagaagaacttttaaatggaaaccctaatattgaaattccagatgacgaggaagcacatgtaaagaaggctttaaaaaaggaaaaagagggtcttccaaaggttttgaaaaaagagcacgatgaatttgttaaaaaaatgatggatgagtcagaaacaattaatattccagaggccttacaatatgacacaatgtatgaagatatactcagtaaaggagaaattcgaaaagaaatcaaaaaacaaatacctaatcctacaacatcttttgagagtatatcaatgacaactgaagaggaaaaagtcgacagtactttaaaaagcgaaatgcaaaatcgtgtctctaagccttcttttgatacctggtttaaaaacactaagatcaaaattgaaaataaaaattgtttattacttgtaccgagtgaatttgcatttgaatggattaagaaaagatatttagaaacaattaaaacagtccttgaagaagctggatatgttttcgaaaaaatcgaactaagaaaagtgcaataaactgctgaagtatttcagcagttttttttatttagaaatagtgaaaaaaatataatcagggaggtatcaatatttaatgagtactgatttaaatttatttagactggaattaataattaacacgtagactaattaaaatttaatgagggataaagaggatacaaaaatattaatttcaatccctattaaattttaacaagggggggattaaaatttaattagaggtttatccacaagaaaagaccctaataaaatttttactagggttataacactgattaatttcttaatgggggagggattaaaatttaatgacaaagaaaacaatcttttaagaaaagcttttaaaagataataataaaaagagctttgcgattaagcaaaactctttactttttcattgacattatcaaattcatcgatttcaaattgttgttgtatcataaagttaattctgttttgcacaaccttttcaggaatataaaacacatctgaggcttgttttataaactcagggtcgctaaagtcaatgtaacgtagcatatgatatggtatagcttccacccaagttagcctttctgcttcttctgaatgtttttcatatacttccatgggtatctctaaatgattttcctcatgtagcaaggtatgagcaaaaagtttatggaattgatagttcctctctttttcttcaacttttttatctaaaacaaacactttaacatctgagtcaatgtaagcataagatgtttttccagtcataatttcaatcccaaatcttttagacagaaattctggacgtaaatcttttggtgaaagaatttttttatgtagcaatatatccgatacagcaccttctaaaagcgttggtgaatagggcattttacctatctcctctcattttgtggaataaaaatagtcatattcgtccatctacctatcctattatcgaacagttgaactttttaatcaaggatcagtcctttttttcattattcttaaactgtgctcttaactttaacaactcgatttgtttttccagatctcgagggtaactagcctcgccgatcccgcaagaggcccggcagtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcatgc

Polypeptide and Protein Sequences

In this module, you can find the polypeptide and amino acid sequences in the entire project. You can quickly locate the sequence you are looking for based on the name of the polypeptide or protein.

Sequence Name Function Sequence (5'→3')
CP-Histag-CP A recombinant dimer expressing MS2 coat protein with His tag ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGGTHHHHHHGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY
CP-GBP3.1-CP A recombinant dimer of MS2 coat protein with GBP3.1 localization peptide ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYTCSKKYPRSPCMGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY
CP-GBP3.1-CP-TAT-Histag A recombinant dimer of MS2 coat protein with GBP3.1 localization peptide, TAT cell-penetrating peptide, and His tag ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYTCSKKYPRSPCMGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYQLQGGPAPGGSAYGRKKRRQRRRLEHHHHHH
EGFP An enhanced green fluorescent protein used for tracing the function of GBP3.1 and TAT peptides MVSFKSLLVLCCAALGAFATKRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
GBP3.1-EGFP-TAT-Histag Enhanced green fluorescent protein fused with GBP3.1 localization peptide, His tag, and TAT cell-penetrating peptide TCSKKYPRSPCMGGGGSGGGGSGGGGSGGGGSMVSFKSLLVLCCAALGAFATKRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKLQGGPAPGGSAYGRKKRRQRRRLQGGPAPGGSALEHHHHHH
TAT Cell-penetrating peptide YGRKKRRQRRR
GBP3.1 Aphid gut localization peptide TCSKKYPRSPCM

Conventional PCR Primer Sequences

In this module, you can find the primer sequences used in PCR throughout the entire project. You can quickly locate the primer you are looking for based on the name of the amplified gene or the species name.

Amplified Gene or Sequence Name Species Primer Name Primer Sequence (5'→3') Product Length (bp) Application
Triple fusion gene dsRNA & glufosinate resistance gene Metarhizium anisopliae pBar-3×dsRNA-F gcccttcctccctttatttc 711bp & 955bp Used for screening successfully transformed Metarhizium after T-DNA insertion via Agrobacterium-mediated transformation
pBar-3×dsRNA-R ctgacatcgacaccaacgat
SacB Promoter, RBS, pchB, pchA, BSMT1 genes Bacillus subtilis BS-F GACCTCGTTTCCACCGGAAT
BS-R TCTTCTTCCGTGATTCCTTG
CP-Histag-CP Recombinant protein CP F CGTACGTGGTACCGGTATG 353bp
CP R AGCAGCCTAGGACATGGGTA
CP-GBP3.1-CP-TAT-Histag Recombinant protein CP F CGTACGTGGTACCGGTATG 353bp
CP R AGCAGCCTAGGACATGGGTA
GBP3.1-EGFP-Histag-TAT Recombinant protein E2 F CCACTACCAGCAGAACACTCC 139bp
E2 R AGCGGTCACGAACTCCAAC
EGFP Recombinant protein E4 F AGTCGCTGCTGGTCTTATGC 371bp
E4 R GTAGTTGCCGTCGTCCTTGA

RT-PCR and qPCR Primer Sequences

In this module, you can find the RT-PCR and qPCR primer sequences used in the entire project. You can quickly locate the primer you are looking for based on the name of the amplified gene or the species name.

Amplified Gene Name Species Primer Name Primer Sequence (5'→3') Product Length (bp) Application
CP19 Toxoptera citricida TCiCP-3-F TTCGATTACGCGGTTCACGA 87 qPCR for amplifying CP19 cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CP19 gene
TCiCP-3-R GTACGCTCCCTTGACCACAT
EF1α Toxoptera citricida TCiEF1α-2-F ACCTGCGAACATCACCACTG 72 qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CP19 gene
TCiEF1α-2-R CCGGGAACAGCTTCTACCAA
CHS Toxoptera citricida TCiCHS-9-F CAACGGCAAAAGACCCGAAC 182 qPCR for amplifying CHS cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CHS gene
TCiCHS-9-R GCTTGCATGGATCGTCTCCT
EF1α Toxoptera citricida TCiEF1α-14-F TGCTGTTGCTTTCGTTCCCA 176 qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CHS gene
TCiEF1α-14-R CCTTGTCAGTTTGGCGACTG
CYP450 Toxoptera citricida TCiCYP-14-F ATCGACACGGCCACGTATTC 121 qPCR for amplifying CYP450 cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CYP450 gene
TCiCYP-14-R CCGAAAACACGCACTCCTCT
EF1α Toxoptera citricida TCiEF1α-5-F ACTGACAAGGCTCTCCGTCT 149 qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CYP450 gene
TCiEF1α-5-R TCAACACGACCTACTGGGAC
Triple fusion gene dsRNA Metarhizium anisopliae pbar-jjz-F GGCCGATCGAGATAAAGAACCA 80 qPCR for amplifying and evaluating dsRNA production efficiency in Metarhizium
pbar-jjz-R TTTCTTCGTGGGTTTCGTGC
Click to EXPAND the References

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Description Description
Design Design
Results Results
Proof of Concept Proof of Concept
Parts collection Parts collection