Use of GAPC gene in enhancing disease resistance of plants
A disease resistance and plant technology, applied in plant products, genetic engineering, plant gene improvement, etc., can solve problems that need to be strengthened, and achieve the effects of no impact on growth and development, good silencing effect, and good repeatability
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Embodiment 1
[0038] The inventors used the methods of co-immunoprecipitation and mass spectrometry to screen the total protein of tobacco with ATG3, the key protein in the autophagy process of Nicotiana benthamiana (Nb for short), as bait, in order to find proteins that may bind to ATG3. The technical idea is mainly to use the expression vector of NbATG3-GFP to express the NbATG3-GFP fusion protein in tobacco leaves. At this time, ATG3 will bind to the protein that interacts with it in vivo, and then through co-immunoprecipitation to screen for possible interactions with the protein. ATG3-bound protein. Specific steps are as follows:
[0039] (1) Take GFP- _A beads 30μl, washed three times with 500μl IP buffer (10% (v / v) glycerol, 25mM Tris pH 7.5, 150mM NaCl, 1×protease inhibitor cocktail (Roche), 0.15% (v / v) NP-40), 4℃ Centrifuge at 1000g for 2min;
[0040] (2) After expressing NbATG3-GFP in tobacco leaves for about 60 hours, extract the total protein of the leaves, mix 1ml protein e...
Embodiment 2
[0047] Autophagy is involved in the regulation of HR cell death and affects plant immune response, so the inventors speculate that GAPC will affect plant disease resistance by affecting autophagy. Therefore, the inventors used VIGS technology to silence GAPC in tobacco to study whether gene silencing affects the growth and development of plants, and the disease resistance of the silenced plants. details as follows:
[0048] 1. Silencing GAPC in N-transgenic tobacco
[0049] The inventors silenced GAPC in N-transgenic tobacco (N-transgenic tobacco seeds were obtained from the lab of Dinesh Kumar, Yale University). The specific method is:
[0050] 1-1 Provide vector pTRV1, pTRV2
[0051] The vectors pTRV1 (GenBank: AF406990) and pTRV2 (GenBank: AF406991.1) are provided. The two vectors are from the laboratory of Dinesh Kumar, Yale University. For vector information, see Liu Y, Schiff M, Marathe R, et al. Tobacco Rar1, EDS1 and NPR1 / NIM1like Genes are required for N-mediated ...
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