Gene GmAP5 for improving disease resistance of plants and use of gene GmAP5
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A gene and transgenic technology applied to the gene GmAP5 for improving plant disease resistance and its application field
Active Publication Date: 2020-12-29
NANJING AGRICULTURAL UNIVERSITY
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However, this type of disease resistance protein is highly specific and can
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Embodiment 1
[0038] 1) Preservation and cultivation of test plants and test strains
[0039] (1) The tested soybean seeds are Williams 82.
[0040] (2) Escherichia coli (E.coli) strain JM109 and Agrobacterium tumefaciens strain K599 are preserved in our laboratory.
[0041] (3) Phytophthora sojae strain P6497 was a gift from Professor Brett M. Tyler (Oregon State University, USA) (the prior art has been published), and is preserved by our laboratory.
[0042] 2) GmAP5 gene cloning:
[0043] (1) Using the soybean genome as a template, using the gene primer F / R of GmAP5 to amplify the full-length GmAP5 gene;
[0044] pBin-GmAP5-GFP-F: gaacgatagggtacccccgggATGCCTCCATCATCACTGTTTTC (SEQ ID No. 4)
[0045] pBin-GmAP5-GFP-R: catggatccgtcgaccccgggCTACTTAGCCAATCTACTACAATCAGCC (SEQ ID No. 5).
[0046] (2) The 50 μL reaction system is: 10 μL of 5× buffer, 4 μL of 2.5mM dNTPs, 0.5 μL of Takara PrimerSTARTaq enzyme, 1 μL of template cDNA, add water to 50 μL, and the final concentration of front and...
Embodiment 2
[0070] Overexpression and silencing of the GmAP5 gene in soybean:
[0071] Refer to the soybean hairy root transformation method established by Kereszt et al. (A. Kereszt et al., Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nature Protocols 2, 948-952 (2007)).
[0072] The specific operation steps are as follows:
[0073] 1) Sow soybean seeds (susceptible varieties such as Hefeng 47) in moist vermiculite, place them in a greenhouse (25°C, 16h light / 8h dark) for cultivation, after about 7 days, remove the watercress for sterilization in the experiment. At this time, the cotyledon differentiation ability is strong.
[0074] 2) After 6 days of planting beans, pick a single colony of Agrobacterium K599 into liquid LB containing antibiotics (50ug / mL kanamycin and 50ug / mL streptomycin), and culture at 28°C for 24h.
[0075] 3) Cotyledon disinfection: Cut off the cotyledon of soybean with better growth with a blade, treat the cotyledon with 75...
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Abstract
The present invention discloses a gene GmAP5 for improving disease resistance of plants and a use of the gene GmAP5, belongs to the field of plant molecular biology and plant genetic engineering, andrelates to the plant-derived disease-resistant gene GmAP5 and a recombinant expression vector and the use of the gene GmAP5. The gene is derived from soybeans and has a nucleotide sequence shown as SEQ ID NO.1. Overexpression of the gene remarkably promotes disease resistance of soybeans to phytophthora sojae and the gene is an ideal gene for enhancing the disease resistance of the plants. The gene is expressed in soybeans or tobaccos through a genetic transformation method, so that the soybeans or tobaccos obtain resistance to various pathogenic microorganisms, and thus field disease resistance of crops can be improved. The gene GmAP5 can be applied to improvement of disease resistance of breeding of the crops.
Description
technical field [0001] The invention belongs to the field of plant molecular biology and plant genetic engineering, in particular, the invention relates to a gene GmAP5 for improving plant disease resistance and application thereof. Background technique [0002] Oomycetes belong to Stramenopiles, which contain a large number of plant and animal pathogens, which pose a huge threat to agricultural production and ecological environment. Phytophthora sojae (Phytophthora sojae) is an important plant pathogen in oomycetes. It mainly infects soybean rhizomes and causes soybean root rot. It causes about 2 billion US dollars in economic losses to the world every year and seriously threatens soybean production. Phytophthora soybean root rot was first introduced into China in the late 1980s, and then spread rapidly in most parts of China, and became one of the main diseases in the main soybean producing areas of Northeast China and Fujian. At present, the pathogen is distributed in mo...
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