1. Preparation of LB (Luria-Bertani) culture medium

Materials:

1. Sodium Chloride 5g
2. Conical flasks
3. Distilled water 500ml
4. Yeast Extract 2.5g
5. Tryptone 5g

Procedures:

Liquid medium:

1. Add 5 grams of Tryptone, 2.5 grams of Yeast extract, and 5 grams of sodium chloride into the conical flask.
2. Add 500 mL of distilled water to the flask
3. Sterilization at 121℃ for 10-15 minutes

1.1 Preparation of Kanamycin

Materials:

1. 0.5 mg kanamycin sulfate

2. 10 ml distilled water

3. 15 ml Centrifuge Tube

Procedures:

1. Add 0.5 mg kanamycin sulfate to the centrifuge tube

2. Add 10 mL of distilled water to the tube

3. Use a syringe and a sterile syringe filter for filtration. 1 ml I each centrifuge tube.

1.2 Inoculation of glycerol bacteria

Procedure:

1. Add 100 μL kanamycin to 100 mL LB culture medium
2. Pour 5 mL of the mixture of LB culture medium and kanamycin into the tube
3. Add 50 μL glycerol bacteria to each 8 separate centrifuge tube.
4. Repeat twice for each of the glycerol bacteria.
5. Pour 5 mL of the mixture of LB culture medium and kanamycin into the tube
6. Put the tubes into the shaking incubator at 37°C and 220 RPM, set for 12-16 hours

1.3 Plasmid extraction

Material:

1. Buffer P1

2. Buffer P2

3. Buffer P3

4. Buffer PW

5. Buffer PW 2

6. Elution Buffer

Procedure:

1. Separate the inoculated glycerol bacteria into centrifuge tubes, each 1.5 ml.

2. Centrifuge the tubes at 10000 rpm for 1 minute

3. Pour out the liquid and centrifuge at 10000 rpm for 10 seconds to make sure the LB culture medium is poured out thoroughly

4. Add 250 μL of Buffer P1 to one centrifuge tube of each type of glycerol bacteria. Stir the bacteria with Buffer P1.

5. Add the mixture to the other 5 centrifuge tubes and mix.

6. Add 250 μL Buffer P2. Stir 8-10 times

7. Add 350 Buffer P3. Shake 8-10 times.

8. Centrifuge the tubes at 12000 rpm for 10 minutes.

9. Use a pipette to transfer liquid to the DNA collection tubes

10. Centrifuge at 12000 rpm, 10minutes.

11. Add 600 μL of Buffer PW2to each tube

12. Centrifuge at 12000 rpm for 1 minute.

13. Repeat steps 11 and 12.

14. Add the mixture to a new 1.5 ml DNA collection tube.

15. Still 2 minutes. Centrifuge at 12000 rpm for 1 minute for elution of DNA

2. Construction of E. coli Expression Plasmids

2.1 PCR amplification of target genes

Material:

1. Te

2. DNA Primer 1

3. DNA Primer 2

4. 2×Hieff PCR Master Mix

5. Water

6. PCR machine

7. Procedure:

1. Add 2 μL plasmid

2. Add 2 μL DNA Primer 1, 2 μL DNA Primer 2, and 2μL 2×Hieff PCR Master Mix

3. Add 19 μL of water to the mixture

4. Mix the things

5. Set PCR to following setting

Step	Temperature (°C)	Time	Cycles
Initial Denaturation	94	5 minutes	1
Denaturation	94	30 seconds
Annealing	50 – 60*	30 seconds	35 Cycles
Extension	72	30-60 sec/kb**
Final Extension	72	10 minutes	1

* Annealing Temperature: Try a temperature 2–5°C lower than the calculated melting temperature (Tm) of your primers.

2.2 Agarose gel electrophoresis AGE「

Material:

• Agarose powder

• TAE Buffer

• DNA Samples

• DNA Ladder

• Gel Box & Lid

• Gel Tray & Combs

• Power Supply

• Microwave or Hot Plate

• UV Light Box

• Pipette and Tips

2.3 Preparing the Gel

1. Weigh 0.5g Agarose Gel powder and 50 mL TAE Buffer

2. Carefully heat the flask in a microwave for 30-60 seconds. It will be very hot!

3. Swirl the flask gently and heat in short bursts until the liquid is completely clear with no floating bits.

4. Let the melted agarose sugar cool for 60 seconds then pour the liquid into prepared gel tray.

5. Set the gel for 20-30 minutes

2.4 Setting Up and Running the Gel

1. Place the gel in the box. Wells should be at the negative side.

2. Pour the TAE buffer into the box that barely covers the gel

3. Use a pipette to suck up the DNA sample with the loading sample.

4. Put it into the wells

5. Set the gel for 20-30 minutes

6. Put on the lids, and the lids should be plugged into the power supply.

7. Turn on the machine and let it run for 20-40 minutes

2.5 Visualize the DNA

1. Turn off the power supply and carefully take the gel out

2. Put the gel into the blue UV light. Bright band is where the DNA is

2.6 Enzyme-digested vector

Materials:​​

1. Purified pET-28a plasmid DNA (100-500 ng/μL)

2. BamHI restriction enzyme (e.g., from NEB)

3. 10x CutSmart Buffer (or compatible buffer)

4. Nuclease-free water

5. Thermostatic water bath or PCR machine

Component	Volume (μL)	Amount
Nuclease-free water	to 20 μL	-
10xCutSmart Buffer	​2.0​	​1x​
pET-28a plasmid DNA	​X​	​500 ng​
BamHI	​1.0​	10-20 units​

2.7 Homologous recombination

Materials:

1. CloneExpress Mix (2×)

2. PCR products of NAS (wild type and mutants), MtnN, and AdeD

3. Linearized pET28a backbone

Procedure:

1. Measure the DNA concentration of gel-purified fragments (NAS variants, MtnN, AdeD).

2. Prepare the recombination system in PCR tubes for the construction of pET28a-NAS:

• 5 μL CloneExpress Mix

• 2.5 μL linearized pET28a backbone

• 2.5 μL NAS variant fragment (wild type or mutant)

3. Prepare additional recombination systems in PCR tubes for the construction of pET28a-MtnN and pET28a-AdeD:

• 5 μL CloneExpress Mix

• 2.5 μL linearized pET28a backbone

• 2.5 μL MtnN or AdeD fragment

4. Place PCR tubes into a PCR thermal cycler and perform ligation reaction at 50 °C for 30 minutes.

2.8 Transformation into competent E. coli cells

Materials:

1. Ligated recombination plasmid

2. Liquid LB solution without antibiotics

3. LB Agar plates with Ampicillin and spectinomycin antibiotics

Procedures:

1. Add recombination plasmid into E.Coli BL21 strain;

2. Placed on ice for 20 minutes;

3. Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;

4. Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;

5. Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;

6. Apply bacterial liquid to LB Agar plates with Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.

2.9 Colony PCR

Material：

1. Plasmid

2. DNA Primer 1

3. DNA Primer 2

4. 2×Hieff PCR Master Mix

5. Water

6. PCR machine

Procedure：

1. Add 2 μL bacteria

2. Add 2 μL DNA Primer 1, 2 μL DNA Primer 2, and 2μL 2×Hieff PCR Master Mix

3. Add 19 μL of water to the mixture

4. Mix the things

5. Set PCR to following setting

Step	Temperature (°C)	Time	Cycles
Initial Denaturation	94	5 minutes	1
Denaturation	94	30 seconds
Annealing	50 – 60*	30 seconds	35 Cycles
Extension	72	30-60 sec/kb**
Final Extension	72	10 minutes	1

* Annealing Temperature: Try a temperature 2–5°C lower than the calculated melting temperature (Tm) of your primers.

2.10 Seed Culture

Material:

• Positive Bacterial Colony

• Liquid LB Medium

• Antibiotic

• Sterile Culture Tubes

• Sterile Pipette Tips and a pipette that can measure 500-1000 µL.

• Incubator Shaker

Procedure:

1. Label Tube

2. Using a sterile pipette tip, add the 5 μL antibiotic to the tube.

3. Add 5 mL of sterile liquid LB medium into the tube.

4. Secure the lid and put it in the incubator shaker set 37°C, 220 rpm for 12-16 hours.

2.11 Scale-Up Culture

Material:

• Seed Culture

• Fresh Liquid LB Medium

• Antibiotic

• Sterile Erlenmeyer Flasks

• Sterile Pipettes or pipette tips

• ncubator Shaker

• Spectrophotometer

Procedure:

1. Label flask

2. Prepare 100 mL LB cultural medium

3. Use a sterile pipette to suck 1 mL seed culture liquid into the medium

4. Put the flask in the incubator shaker, speed 220 rpm, temperature 37°C for 2 hours

3. Protein Expression and Purification

3.1 Preliminary Experiment for Protein Expression

3.1.1 Amplification Culture for Correct Colony

Goal: To obtain sufficient amounts of target NAS mutant proteins for SDS-PAGE verification.

Materials:

1. Liquid LB medium

2. NAS mutant plasmids and corresponding cofactor expression constructs (e.g., AtNAS1-Wildtype, AtNAS1-Truncated, AtNAS1-TRG_to_AAA, and cofactor-related vectors)

3. Ampicillin and spectinomycin antibiotics

4. Inducer: IPTG

5. Iso-thermic shaker

Procedures:

1. Inoculate 1000 μL of bacterial culture containing NAS mutant plasmids and cofactor plasmids into 100 mL of liquid LB medium, and add 100μL of both Ampicillin and spectinomycin antibiotics.

2. Incubate the culture in an iso-thermic shaker at 37℃ for ~3 hours until the absorbance at OD600 reaches 0.4–0.6.

3. Add IPTG to the culture at a final concentration of 1 mM to induce protein expression.

4. Continue incubation at 37℃ for 6 hours, and collect samples for subsequent analysis.

3.1.2 Protein Crude Extraction and SDS-PAGE

Goal: To confirm the expression of the desired NAS mutant proteins.

Materials for Protein Crude Extraction:

1. 500 μL of bacterial culture from different induction conditions

2. Centrifuge

3. Double distilled water (ddH₂O)

4. 6×Protein loading buffer

3.1.3 Procedures for Protein Crude Extraction

1. Centrifuge the samples at 12,000 rpm for 1 minute.

2. Discard the supernatant and resuspend the cell pellet in 50μL of 1×Protein loading buffer diluted with ddH₂O.

3. Heat the resuspended samples at 95℃ for 15 minutes using a PCR thermal cycler.

Materials for SDS-PAGE:

1. 12.5% SDS-PAGE Color Preparation kit (Sangon)

2. Electrophoresis buffer (Tris-Glycine)

3. Protein ladder

4. 6×Protein loading buffer

5. Vertical electrophoresis system

6. Coomassie blue staining solution

Procedures for SDS-PAGE:

1. Mix 2.2 mL 2×separating gel solution, 2.2 mL 2× separating gel buffer, and 44 μL catalyst in a centrifuge tube for preparing the separating gel.

2. Pour the mixture slowly into the casting frame to avoid bubble formation.

3. Overlay with 1 mL ddH₂O and allow the separating gel to solidify for ~8 minutes.

4. Remove the water carefully after polymerization.

5. Mix 825 μL 2× stacking gel solution, 825 μL 2× stacking gel buffer, and 11 μL catalyst for stacking gel preparation.

6. Pour stacking gel mixture on top of the separating gel, insert comb gently to avoid air bubbles, and allow to solidify for ~12 minutes.

7. After polymerization, carefully remove the comb and wash the wells with electrophoresis buffer.

8. Load 10 μL of protein ladder into the first well, followed by sample loading into successive wells.

9. Place the gel into the vertical electrophoresis system.

10. Run electrophoresis at 120 V for ~90 minutes.

11. Stain the gel with Coomassie blue for 10 minutes, then destain repeatedly until the background becomes clear and the protein bands are visible.

3.2 His-Tag Affinity Purification of Proteins

Protein Purification Using a Nickel Column (Ni-NTA)

Materials:

• Bacterial Pellet

• Lysis Buffer

• Equilibration/Wash Buffer

• Elution Buffer

• Ni-NTA Resin

• Empty Chromatography Column

• Centrifuge and Tubes

• Ice Bucket

Procedure:

1. Grow a bacterial culture expressing the NAS mutant plasmid with cofactors to an OD600≈0.6, induce with IPTG (final concentration 1 mM), and incubate at 16℃ for 12 hours.

2. Harvest the cells by centrifugation at 5,000 × g for 15 minutes at 4℃.

3. Resuspend the pellet in lysis buffer (10 mL of buffer per 1 g of wet cell pellet).

4. Add lysozyme (1 mg/mL) and PMSF (1 mM) and incubate on ice for 30 minutes.

5. Disrupt the cells by sonication (2 seconds on, 5 seconds off, for a total of 10 minutes, incubate on ice).

6. Centrifuge the lysate at 12,000 × g for 30 minutes at 4℃ and collect the supernatant (soluble fraction).

7. Equilibrate the Ni-NTA resin with 5 column volumes (CV) of lysis buffer.

8. Ensure the resin is completely resuspended, avoiding the introduction of bubbles.

9. Add the clarified supernatant to the equilibrated Ni-NTA resin.

10. Incubate at 4℃ with gentle shaking for 30-60 minutes to allow binding of the His-tagged NAS protein.

11. Wash the resin with 10 column volumes (CV) of wash buffer (20-40 mM imidazole) to remove nonspecifically bound proteins.

12. Elute the bound His-tagged NAS mutant protein with elution buffer (250 mM imidazole).

13. Collect the eluate (e.g., 1 mL aliquots) and monitor protein elution by measuring absorbance at 280 nm.

4. Functional Tests Part1: Mutation Sites Selection

4.1 Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability

Materials:

• SAM (S-adenosyl-methionine) substrate

• Purified mtnN enzyme

• Purified adaD enzyme

• AtNAS1 protein sample (various mutants: AtNAS1-Wildtype, AtNAS1-Truncated, AtNAS1-TRG_to_AAA, etc.), quantified (mg/mL) and stored on ice

• 50 mM Tris-HCl buffer, pH 8.7 (fresh or filter-sterilized)

• ddH₂O (purified deionized water)

• 1× PBS or a suitable dilution buffer

• Appropriate protein dilution buffer (if diluting the protein to 0.15 mg/mL)

• BioTek microplate reader

• Multichannel pipette and filter tips

• Ice-cold box, centrifuge

• Protein quantification reagent (Bradford/BCA)

• Sterile microcentrifuge tubes, vortexer, timer

• Septum film or cover plate

Procedure:

1. Prepare a reaction mixture containing 1 mg/mL mtnN, 1 mg/mL adaD, 50 mM Tris-HCl buffer (pH 8.7), and 0.15 mg/mL purified NAS1 mutant protein.

2. Preincubate the mixture at 37℃ for 3 minutes.

3. Initiate the enzymatic reaction by adding 0.125 mM SAM substrate and gently mix five times.

4. Immediately add the reaction mixture to a 96-well microplate.

5. Place the microplate in a BioTek microplate reader, set the temperature to 37℃ and the wavelength to 265 nm, and measure in kinetic mode (continuous reading).

6. Record the absorbance change over time for each well, using five technical replicates for each NAS1 mutant protein.

7. Calculate the initial reaction rate using the Lambert-Beer law and the molar extinction coefficient of adenine (ε = 6700 M⁻¹·cm⁻¹ at 265 nm).

8. Enzyme activity was normalized to protein concentration and expressed as nkat/mg protein.

9. Enzyme activity of NAS1 mutants was compared with that of wild-type AtNAS1 and truncated AtNAS1 proteins to identify the most potent variant.

4.2 Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates

Materials:

1. Computer workstation with internet access and sufficient computational performance.

2. Online databases and software:

• UniProtKB (for protein sequence acquisition)

• SWISS-MODEL (for homology modeling and PDB structure prediction)

• Small molecule databases (e.g., PubChem, ZINC, or ChEMBL; for ligand acquisition in SDF format)

• Discovery Studio (DS) software (for ligand optimization and molecular modeling)

• PyMOL (for protein visualization and preprocessing)

• AutoDock Tools (ADT) (for receptor/ligand preparation, pdbqt conversion, and grid box setup)

• AutoDock Vina or AutoDock 4.2 (for molecular docking)

3. Standard computational chemistry resources:

• Force fields and energy minimization tools integrated in DS or AutoDock.

• Visualization software for docking results (DS, PyMOL).

Procedure:

1. Protein sequence acquisition and modeling

• Search the target gene in UniProtKB and download its protein sequence in FASTA format.

• Upload the sequence to SWISS-MODEL to perform homology modeling.

• Select the model with the highest sequence identity and model quality.

• Download and save the resulting protein structure as PDB format for later use.

2. Ligand acquisition and optimization

• Search the target small molecule in a chemical database (e.g., PubChem or ZINC).

• Download the ligand in SDF format.

• Open the ligand in DS software, perform energy minimization to optimize its geometry, and save the optimized structure in PDB format.

3. Protein preprocessing

• Load the protein PDB file into PyMOL.

• Remove water molecules and any non-relevant ligands or ions.

• Save the cleaned protein structure as PDB format.

4. Receptor preparation

• Import the protein into AutoDock Tools (ADT).

• Add polar hydrogens and assign Gasteiger charges.

• Save the processed protein as pdbqt format, defined as the receptor.

5. Ligand preparation

• Import the optimized ligand into ADT.

• Add hydrogens, assign charges, and define rotatable bonds.

• Save the ligand structure as pdbqt format, defined as the ligand.

6. Grid box setup

• Since the binding pocket is unknown, define the grid box to cover the entire protein structure.

• Save the grid parameter file for docking.

7. Docking simulation

• Perform docking using AutoDock.

• Run at least 50 independent docking simulations to ensure reliability and reproducibility of results.

8. Result analysis

• Analyze docking outputs and compare binding energies.

• Select the conformation with the lowest binding energy and the highest clustering frequency as the optimal binding pose.

• Visualize docking results using DS or PyMOL, displaying the 3D binding conformation, protein surface interaction, and binding pocket environment.

4.3 Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins

Materials:

1. Software and computational resources

• GROMACS 2025 molecular dynamics simulation package

• ACPYPE or AmberTools (for ligand topology generation with GAFF2 force field)

• VMD, PyMOL, or Chimera (for visualization of trajectories and results)

• g_mmpbsa or GMXPBSA tool (for MM-PBSA calculations)

• High-performance computing (HPC) cluster or workstation with sufficient CPU/GPU resources

2. Force fields and parameters

• AMBER99SB-ILDN force field (for protein)

• GAFF2 force field (for ligand parameterization)

• TIP3P water model (for solvation)

3. Input files

• Protein structure file (PDB format, pre-processed and minimized)

• Ligand structure file (optimized and converted into topology using GAFF2)

Procedure:

1. System setup

• Import the protein structure into GROMACS.

• Generate topology for the protein using the AMBER99SB-ILDN force field.

• Generate ligand topology and parameters using GAFF2 (via ACPYPE/AmberTools).

• Merge the protein and ligand topology files, checking for atom type compatibility and resolving conflicts.

2. Define simulation box

• Place the protein–ligand complex in a cubic box.

• Ensure the minimum distance from the solute surface to the box boundary is 1.2 nm.

3. Solvation and ion addition

• Solvate the system with TIP3P water model.

• Neutralize the system and adjust ionic concentration to 0.15 M NaCl, mimicking physiological conditions.

4. Energy minimization

• Perform steepest descent energy minimization to remove steric clashes.

• Ensure the maximum force on the system is below the threshold (e.g., <1000 kJ/mol/nm).

5. Equilibration

• Conduct NVT equilibration (constant number of particles, volume, and temperature) for 1,000,000 steps (~2 ns) at 310 K.

• Conduct NPT equilibration (constant number of particles, pressure, and temperature) for 1,000,000 steps (~2 ns) at 310 K and 1 bar.

• Use coupling constants of 0.1 ps for temperature and pressure.

6. Production molecular dynamics simulation

• Perform NPT production MD for 100 ns.

• Simulation parameters:

• Time step: 2 fs

• Number of steps: 50,000,000

• Temperature: 310 K (using Nose-Hoover thermostat)

• Pressure: 1 bar (using Parrinello-Rahman barostat)

• Periodic boundary conditions (PBC) applied in all directions

• Save trajectory data every 1000 steps for later analysis.

• 7. Trajectory analysis

• Calculate RMSD (root mean square deviation) to evaluate structural stability.

• Calculate RMSF (root mean square fluctuation) to assess residue-level flexibility.

• Measure radius of gyration (Rg) to monitor molecular compactness.

• Compute solvent accessible surface area (SASA) to evaluate solvent exposure.

• Monitor the number of hydrogen bonds between protein and ligand over time to assess interaction stability.

8. Free energy calculations

• Construct free energy landscapes (2D and 3D FELs) based on principal components or RMSD/Rg coordinates.

• Perform MM-PBSA analysis using g_mmpbsa/GMXPBSA:

• Compute the average binding free energy between protein and ligand.

• Perform per-residue energy decomposition to identify key residues contributing to binding affinity.

9. Result visualization and reporting

• Use PyMOL/VMD for trajectory visualization.

• Present energy profiles, RMSD/RMSF plots, Rg, SASA, hydrogen bond occupancy, and FEL graphs.

• Summarize MM-PBSA results in tables and highlight critical binding residues.

5. Functional Tests Part2: Plant Model Validation

5.1 Construction of N. benthamiana Transient Expression Plasmids

The method of this step is similar to 2.7, except that the vector and gene are different.

5.2 Transient N. benthamiana Expression Assay

5.2.1 Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy

Materials:

• Expression vector

• Control vector

• Competent cells of the Agrobacterium tumefaciens strain (GV3101) for transient expression.

• LB medium and antibiotics for Agrobacterium culture.

• Infiltration buffer: 10 mM MgCl2, 10 mM MES pH 5.6, 100 µM acetosyringone.

• 4-6 leaf Nicotiana benthamiana (or tobacco) plants for Agrobacterium-mediated transient expression.

• Syringe (without needle) for leaf infiltration.

• Sterile microcentrifuge tubes, pipettes, and tips.

• Glass slides, coverslips, vacuum grease or gaskets, and mounting medium.

• Fluorescent organelle stain.

• Confocal laser scanning microscope.

• Image analysis software (ImageJ).

Procedure:

1. Construct Preparation

2. Agrobacterium Transformation and Cultivation

3. Transform the construct into Agrobacterium (GV3101) and select transformants on appropriate antibiotic plates.

4. Pick a single colony and culture in LB medium containing antibiotics at 28°C with shaking overnight.

5. The next day, subculture the cell into fresh LB medium and grow to an OD600 of approximately 0.6–1.0.

6. Prepare the Infiltration Suspension

7. Resuspend the pellet in infiltration buffer and adjust the final OD600 of each construct to approximately 0.3–0.5.

8. If not already added, add acetosyringone to a final concentration of 100 µM.

9. Incubate the suspension at room temperature for 2–3 hours to activate the Agrobacterium virulence genes.

10. Using a needleless syringe, infiltrate the abaxial surface of the selected Nicotiana benthamiana leaf with the Agrobacterium suspension.

11. Mark the infiltrated area and maintain the plant under normal growth conditions (22-25°C, 8 hours of darkness).

12. Allow expression for 48-72 hours.

13. Sample preparation for imaging

14. At the selected time point, excise a small leaf disc from the infiltrated area.

15. Place the leaf disc, abaxially facing down, on a glass slide in a drop of water or mounting medium.

16. Use spacers or vacuum aspiration to avoid squeezing the tissue and cover with a coverslip.

17. Confocal imaging

18. Image processing and analysis

5.2.2 Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using Real-time Quantitative PCR In Vivo

Materials:

1. Plants expressing wild-type and mutant AtNAS1 or leaf samples transiently expressed via Agrobacterium, and corresponding controls (empty vector/wild-type).

2. Disposable scissors and forceps, quick-freezing equipment (liquid nitrogen or dry ice box), and an RNase-free reagent environment.

3. RNA extraction reagents/kits

4. Reverse transcription reagents/kits: Reagents or kits used to reverse transcribe total RNA into cDNA.

5. qPCR primers: Specific primers for target iron cycle-related genes and internal reference genes.

6. Real-time fluorescence quantitative PCR reagents

7. RNase-free pipette tips, RNase-free centrifuge tubes, 96/384-well qPCR reaction plates, and optical cover film.

8. Real-time qPCR instrument

9. Analysis software for Ct value processing, ΔΔCt calculation, graphing, and statistical testing.

Procedure:

1. Collect similar tissues from each treatment group at the designated time points.

2. Rapidly process and immediately freeze to preserve RNA integrity.

3. Extract total RNA from frozen tissues and perform DNA removal.

4. Reverse transcribe the quality-controlled RNA into cDNA using an appropriate reverse transcription reagent.

5. qPCR primer design.

6. qPCR reaction preparation.

7. qPCR run and raw data collection.

8. Data processing and relative quantification.

9. Statistical analysis and result interpretation.

5.3 Construction of B. subtilis Expression Plasmids

The method of this step is similar to 2.7, except that the vector and gene are different.

5.4 Biofortification Experiment of Soil-Based N. benthamiana Cultivation Using B. subtilis Stably Expressing AtNAS1 Mutated Proteins

5.4.1 Effect of B. subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and Biomass

Materials:

1. N. benthamiana seedlings for soil cultivation.

2. Bacillus subtilis strains stably expressing AtNAS1 variants (wild-type and mutant) and a control strain.

3. Sterile or specialized potting soil suitable for N. benthamiana.

4. Growth chamber with controlled light, temperature, and humidity.

5. Drying oven for handling and measuring fresh and dry biomass.

Procedure:

1. Grow Nicotiana benthamiana until it reaches a growth stage suitable for microbial treatment.

2. Apply a strain of Bacillus subtilis to the plant rhizosphere, maintaining a control treatment.

3. Regularly observe plant growth, developmental changes, and health.

4. Collect plant growth data, including fresh and dry biomass.

5.4.2 Effect of B. subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco Leaves Using ICP-MS

Materials:

1. Nicotiana benthamiana leaves harvested from soil

2. Bacillus subtilis strains stably expressing AtNAS1 variants and a control strain

3. Leaf harvesting tools, a desiccator or freeze dryer for tissue dehydration, and a mortar and pestle for grinding dried tissue

4. Acids and buffers suitable for trace element digestion, compatible with ICP-MS instrumentation

5. ICP-MS instrument

6. Digestion tubes

7. Volumetric flasks

8. Sterile tweezers

9. Sample labeling material

Procedure:

1. Define treatment groups (e.g., uninoculated, parental B. subtilis, B. subtilis expressing wild-type AtNAS1, and each AtNAS1 mutant) and ensure sufficient biological replicates for statistical comparisons.

2. Leaf Sampling: Collect comparable leaves from each plant to avoid contamination; label and record sample metadata.

3. Tissue Drying and Homogenization: Dry leaves using standard methods and then grind into a fine, uniform powder for consistent analysis.

4. Sample Digestion: Digest the powdered tissue with acid or other suitable reagents to release trace elements into solution.

5. ICP-MS Measurement: Analyze the digested samples using ICP-MS to determine iron and zinc concentrations.