Soybean disease-resistant gene and its coded protein and uses
A disease-resistant gene, soybean technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of rapid mutation of pathogenic bacteria, limited role of conventional breeding, and lack of antigens.
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Embodiment 1
[0043] Embodiment 1, the cloning of GmeR
[0044] plant material:
[0045] For the soybean variety "Kehe" No. 1, when the seedlings were 4 weeks old, the whole plant was sprayed with 2.0mM salicylic acid for 48 hours.
[0046] 1. Primer design for GmeR 5’Race
[0047] According to the muskmelon eR gene sequence published in the gene bank (www.ncbi.nlm.nih.gov), the sequence alignment was carried out on the Internet, and it was found that there was a 465bp DNA in the EST sequence of soybean induced by 2.0mM salicylic acid for 48 hours Fragment, up to 83% homology with the 3' end sequence of the melon eR gene, it is speculated that this sequence may be the 3' end gene sequence of the homologous gene in soybean, and this homologous gene in soybean is named as GmeR, and the The 3'-end PCR primers P1 and P2 of GmeR5'Race, and the 5'-end primers P3 and P4 are provided by the GeneRacer kit (Cat.No.L1502-01) of Invitrogen, and the sequences of the above primers are as follows:
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Embodiment 2
[0118] Embodiment 2, the construction of GmeR plant expression vector pGmeR121
[0119] 1. Construction of intermediate vector pCV121
[0120] The gene GmeR expression cassette constructed in this example includes the following gene expression regulatory elements: 35S promoter at the 5' end, gene GmeR and NOS terminator at the 3' end. First construct the intermediate vector pCV121 without the gene GmeR, the construction process is shown in Figure 5, and the specific process includes the following steps:
[0121] 1. Construction of intermediate vector pUC121
[0122] Plasmid vector pBI 121 (Beijing Baierdi Biotechnology Co., Ltd.) was double-digested with restriction endonucleases Sac I and EcoRI, and the 260 bp T-NOS terminator fragment was recovered, which was double-digested with the same enzymes. The vector pUC19 was ligated to obtain an intermediate vector named pUC121, which was identified by restriction endonuclease Sac I and EcoR I, and the results of enzyme digestion...
Embodiment 3
[0129] Embodiment 3, identification of disease resistance of GmeR transgenic tobacco
[0130] The plant expression vector pGmeR121 constructed in Example 2 was transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. The Agrobacterium tumefaciens LBA4404 transformant integrated with pGmeR121 was then transformed into tobacco NC89 by leaf disc method to obtain 20 GmeR transgenic tobacco strains. Using the isolated leaf inoculation method, with non-transgenic tobacco as a control, the genetically modified tobacco is carried out to identify the resistance of tobacco black shank (Black shank) and bacterial wilt. The specific methods are as follows:
[0131] 1. Identification of tobacco black shank resistance in GmeR transgenic tobacco
[0132] The production method of black shank fungus valley: boil the millet until about half of the grains bloom, remove it, put it into a triangular flask, and sterilize it under high pressure for 1 hour. Inoculate the cultured b...
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