Method for improving drought resistance and herbicide resistance of soybean
A herbicide-resistant soybean technology, applied in the field of plant genetic engineering, can solve the problem of no FvC5SD gene transfer, etc., to achieve the effect of improved herbicide resistance, broad application prospects, and improved drought tolerance
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Embodiment 1
[0026] Example 1 Plant expression vector construction
[0027] According to the nucleotide sequence of the FvC5SD gene (GenBank accession number JN696291.1) published on NCBI, see sequence SEQ ID NO: 1, the FvC5SD gene was artificially synthesized according to the plant codon bias. According to the molecular characteristics of the multiple cloning sites of the plant binary expression vector, an endonuclease SpeI cleavage site was introduced upstream of the FvC5SD gene, and an endonuclease SacI cleavage site was introduced downstream. The full-length sequence of the FvC5SD gene (891bp) was amplified by PCR method, and after SpeI and SacI digestion by the method of double cutting and double joining, the FvC5SD gene was connected to the pYL vector (commercially available) to obtain the expression vector pYL-FvC5SD ( figure 1 A) See sequence SEQ ID NO: 2, the constructed plasmid is detected by sequencing and enzyme digestion ( figure 1 B), the verified correct plant expression v...
Embodiment 2
[0028] Example 2 Genetic Transformation of Soybean Mediated by Agrobacterium
[0029] In this study, the Agrobacterium strain EHA101 carrying the target gene FvC5SD was selected, and the soybean recipient variety Williams82 was genetically transformed using the Agrobacterium tumefaciens-mediated genetic transformation method of soybean cotyledon nodes. The process of Agrobacterium-mediated soybean cotyledon node transformation method is shown in figure 2 , the basic process is as follows:
[0030] (1) After Agrobacterium was cultured at 28°C for 16 hours, the cells were collected and transferred into YEP liquid medium until the OD600 value was 0.5-0.7 for later use;
[0031] (2) Mature seeds of soybean recipient variety Williams82 were selected and sterilized with chlorine gas for 16 hours. After sterilization, the seeds were put into GM germination medium (medium formula refers to OLHOFT, etc.) and germinated under low light for 16 hours;
Embodiment 3
[0038] Example 3 Detection of marker gene test strips of transgenic plants
[0039] Since the marker gene on the plant expression vector pYL used in this study is the herbicide resistance gene bar of Streptomyces hygroscopicus, we can use commercial bar gene test strips (EnviroLogix, USA, catalog number: 010475) as T0 generation Protein immunological detection of marker genes in transgenic seedlings. How to use Take out the test strip, hold the top of the test strip, and make a test mark. Do not remove the protective film. Keeping the test strip vertical, insert the labeled end into the centrifuge tube or extraction bag. The insertion part should not exceed 0.5cm. It remains plugged in throughout the detection process. The quality control line appears within 3-5 minutes, and the longest reaction time is 30 minutes. At this time, the test strip can be taken out. Quality control lines are used to ensure the accuracy of test results. If the control line does not appear, the...
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