Since drought can induce the expression of CLE25 in the root, and can be transferred from the root to the leaf for a long distance to bind to the receptor proteins BAM1 and BAM3, it induces NCED3 expression, resulting in an increase in ABA concentration and a decrease in the degree of stomatal openness in response to plant water shortage. Therefore, we chose the promoter of NCED3, one of the key enzymes in ABA biosynthesis, in response to drought stress.

Meanwhile, the Department of Cell and Developmental Biology at the University of California, San Diego and the State Key Laboratory of Crop Genetics and Germplasm Improvement at Nanjing Agricultural University published a team entitled "A reporter for noninvasively monitoring gene expression and plant transformation" in Horticulture Research on September 19, 2020 A research-oriented article that focuses on a betanoside-based chromogenic reporting system called RUBY. The system turns the plant red without the help of any instruments, and can be observed with the naked eye alone, which is a good visualization system, so we chose RUBY as our reporting system.

In the team's expected results, the plants that received the NCED3promoter-RUBY early warning system would appear red visible to the naked eye under drought stress.

At present, the immune mechanism of plant bodies in response to pest and disease stress is not completely clear, and after reviewing the literature, it is found that glutamic acid, as a signaling substance for plant mechanical damage, increases the concentration of calcium ions in the cytoplasm and conducts to other parts of plants by being perceived by the glutamate receptor-like protein family, thereby activating the systematic defense response of plants in advance. Further studies found that GLR2.9 was involved in the innate immune response of Arabidopsis thaliana and was significantly upregulated during bacterial infection, which was suitable as a receptor in response to biological stress.

In the literature research, it was found that Professor Xiong Aisheng's team from the State Key Discipline of Vegetable Science/State Key Laboratory of Crop Genetics and Germplasm Innovation of Nanjing Agricultural University published a research brief entitled "A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation" online in The Crop Journal. Using the visual color and fluorescence characteristics of betaxanthin, a convenient reporting system Bx was developed and designed, which turned the plants yellow and produced bright green fluorescence under the excitation of blue light.

After designing the two reporting systems, the team intends to cross-combine the promoter and the reporting system, so as to not only obtain a variety of early warning systems, but also make it easier to verify the success of the early warning system. As our first successful system, we decided to use NCED3promoter-RUBY to perform stable transfer treatment on Arabidopsis thaliana plants to obtain stable traits to observe plants. The rest of the system uses teleportation technology to observe the phenomenon on tobacco.

It can be seen that our experiment is divided into two parts: molecular experiment and plant experiment, and two reporting systems are divided into two reporting systems and carried out in parallel.

So in the molecular experiment part, the team was divided into two groups, one group was responsible for the construction of the NCED3promoter-RUBY early warning system, and the other group was responsible for the construction of the GLR2.9 promoter-Bx early warning system.

The molecular experimental part starts with the most basic plasmid construction, and the NCED3/GLR2.9 promoter fragment is obtained in the Arabidopsis whole DNA set, and ligated with the target gene fragment synthesized from the biocompany to construct a complete plasmid.

After obtaining the plasmid, it was transformed into E. coli and compared with the sequencing results to confirm that the target gene and promoter gene were intact.

After the plasmid is determined to be correct, agrobacterium transformation is performed. Among them, NCED3promoter-RUBY was stabilized to obtain stable plants. The rest of the early warning system is instantaneous on the tobacco to see if the reporting system is achieving the desired effect.

After verifying that the early warning system can achieve the expected effect, the transgenic plants are transformed to obtain the final product of the project.

Reference

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