DESCRIPTION

The initial goal of our project is to improve the yields of the crops. We plan to achieve this goal by improving the efficiency of the nitrogen-fixation of plants and defending plants from attacks by nematodes. (The focus of our project is leguminous plants)

In order to improve the nitrogen-fixation efficiency, we chose to use Bacillus subtilis(B.subtilis) to synthesize luteolin, a kind of flavonoid that can help attract nitrogen-fixing bacteria. And because Escherichia coli (E. coli) is simpler, we used E. coli to verify the first half of the pathway from L-Tyr (a substance common in bacteria) to naringin (a precursor of luteolin). We use the Cre-loxP principle to make the process controllable.

In order to defend the plants from attacks by nematodes, we chose to express Cry region proteins in B. subtilis and conduct toxicity experiments on C. elegans. We use a salicylic acid molecular switch to make the plants only produce Cry region proteins when bitten by nematodes.

Nitrogen is a component of various key compounds in plants, and the nitrogen content in soil is one of the core factors affecting plant growth, development, yield, and quality. However, nitrogen deficiency exists in some regions, such as acidic soil areas with abundant rainfall, arid sandy soil areas, and high-altitude cold tundra areas. Up to now, nitrogen deficiency is a global issue, with some countries being particularly severely affected.

In addition, a large number of nematodes in the soil also pose a major threat to plants. Nematodes feed on plant roots, damage root cells, cause impairment to the physical function and structure of plant roots, and thereby affect the absorption of nitrogen. At the same time, nematodes inject auxin-like substances when feeding, which leads to abnormal division and expansion of plant root cells. This squeezes the plant vascular bundles, preventing nitrogen from being transported to the required parts within the plant.

Therefore, our goal is to solve the problem of nitrogen deficiency faced by plants while protecting them from nematode feeding. We know that leguminous plants are the main source of nitrogen enrichment in the soil. Their key mechanism is to secrete flavonoid substances to attract rhizobia, thereby achieving nitrogen fixation. However, in nitrogen-deficient areas, leguminous plants secrete fewer flavonoids and thus cannot effectively attract rhizobia. Therefore, through literature research, we have decided to use the luteolin secreted by B. subtilis to increase the content of flavonoids in the soil. To address the problem of nematode infestation, we plan to utilize the specific proteins secreted by B. subtilis to kill nematodes. B. subtilis is a bacterium widely present in soil, with low nutrient requirements. It can reproduce in nitrogen-deficient soil and efficiently secrete a variety of proteins and metabolites. Therefore, by modifying B. subtilis, we can effectively obtain the target bacterial strain we need.

In the molecular part, we use E. coli to verify the first half of the pathway from L-Tyr to naringin. To do so, we transformed three plasmids::pETDuet-CHS-CHI + pACYCDuet-matB-matC + pCDF-TAL-4CL — into E. coli and used SDS-PAGE and Western Blot measurements to test whether the enzymes were successfully expressed. After that, we used HPLC (High Performance Liquid Chromatography) to test the existence of naringin.

To verify the second half of the pathway from naringin to luteolin, we constructed a plasmid named pBE2R-FNS-F3’H (structure: p43 promoter-FNS-F3’H-terminator) and transformed it into B. subtilis. We then used SDS-PAGE to check for successful enzyme expression and HPLC to detect luteolin production.

In order to achieve precise control over our engineered bacteria, we screened phages targeting the B. subtilis BS168 strain from the environment. After expressing the product in the engineered B. subtilis, we used phages to regulate its gene expression and programmed cell death, thereby terminating the reaction. To further achieve dual regulation of initiation and termination of the engineered bacteria's function, we designed the Cre-loxP recombination system. We inserted a “loxP-terminator-loxP” (LSL) system for promoter activation and used the λ phage to deliver the Cre enzyme into the engineered B. subtilis.

In the nematode part, we chose to use B. subtilis to express Cry5Ba and Cry6Aa proteins, two of the most toxic proteins according to literature. We also expressed a combined Cry5Ba&6Aa toxic protein. After expression, we conducted toxicity experiments on C. elegans and used their mortality rate to visualize the toxicity strength of the different proteins.

To meet the project's requirement for real-time monitoring of soil salicylic acid content, we have developed a rapid testing tool designed for convenient field operation. This tool comprises an external protective sleeve and an integrated detection tube, which contains salicylic acid-specific test paper. For use, the sleeve is inserted into the soil and water added to form an eluate. The detection tube is then inserted to allow the test paper to react with the liquid. The resulting colour change enables semi-quantitative analysis of salicylic acid content, providing critical data support for engineering applications.

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