Gene GmWRKY148 capable of improving resistance to phytophthora sojae and application of gene
A soybean Phytophthora, gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of soybean yield and economic loss, resistant varieties cannot maintain continuous resistance, etc.
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Embodiment 1
[0041] 1) Cloning and bioinformatics analysis of GmWRKY148 gene
[0042] Phytozome (http: / / www.phytozome.net / ) was used to find genes homologous to Arabidopsis AtWRKY44 in soybean with AtWRKY44 (AT2G37260.1) as a probe, and BLAST comparison was performed. As a result, it was found that the number of homologous genes was large, and after the induction of Phytophthora soybean P6497, the gene with a large expression change was selected, namely GmWRKY148, and it was determined as the research object ( figure 1 ). Primers were designed, and the primer sequences are listed in Table 1. The results of three-dimensional structure analysis of GmWRKY148 protein showed that ( figure 2 ), which consists of 7 α-helices and 4 β-sheets. Four β-sheets are typical features of WRKY domain, and the β-strand containing WRKYGQK motif can interact with the 6bp W-box (TTGACY) region. Evolutionary analysis showed that GmWRKY148 belongs to soybean WRKY transcription factor family gene, and is very...
Embodiment 2
[0051] The genetic engineering application of embodiment 2 GmWRKY148
[0052] According to the cDNA sequence of GmWRKY148, primers were designed to amplify the complete coding open reading frame (ORF), and adapter primers were introduced into the primers. For the DNA sequence, the vector is constructed by double restriction digestion, and the restriction sites are Kpn1 and BamH1. The complete ORF was inserted into the expression vector pBINGFP2, and the plant overexpression vector 35S-WRKY148::GFP ( Figure 7 ). It and the empty load were respectively transformed into Agrobacterium rhizogenes K599 by electric shock transformation method, and then the transgenic soybean root hairs were obtained by Agrobacterium-mediated method. Then according to the marker protein GFP on the carrier, the positive soybean root hairs obtained are initially screened using a fluorescent stereomicroscope, and the positive hairy roots are expanded and cultivated on the White medium ( Figure 8 , a...
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