Disease-resistant and drought-enduring protein gene GhSNAP33 from cotton and application of disease-resistant and drought-enduring protein gene GhSNAP33
A cotton and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of lack of disease resistance genes and drought tolerance genes, and achieve the effect of improving plant varieties and improving plant disease resistance.
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Embodiment 1
[0037] Cloning of embodiment 1 cotton GhSNAP33 gene
[0038] 1) The cotton variety in the present invention is from Upland Cotton Zhongzhimian No. 2 (a variety with high resistance to blight and Verticillium wilt approved by my country, which can be used as a suitable material for cloning of resistance to diseases and insect pests). Sow cotton in a 1:1 mixed medium of nutrient soil: vermiculite, keep the nutrient medium moist on the surface of the flowerpot with a plastic film, cultivate at 25°C for one week, remove the film, and continue to cultivate for 2 weeks. Cotton seedling samples were collected, RNA was extracted using the EASYspin Plant RNA Rapid Extraction Kit (Bomed), and stored at -80°C for later use.
[0039] 2) The cDNA sequence of cotton GhSNAP33 gene was screened out by in situ hybridization technique, and positive clone was obtained after enzyme digestion identification, which was identified as a partial sequence of SNAP33 gene. The cotton genome database was...
Embodiment 2
[0044] Induced expression analysis of embodiment 2GhSNAP33 in cotton
[0045] Cultivate 2-week-old cotton seedlings with the method in Example 1, gently remove them from the soil, wash them with clear water, and dry them. Use 1×10 6 Spores / mL Cotton Verticillium dahliae Vd991 infected cotton seedlings, collected 0, 0.5, 12, 24, 72, 120 and 168h samples after infection and preserved them for later use; meanwhile, simulated drought treatment with 10% (w / v) PEG6000, Collect and process 0, 1, 3, 6, 12, 24 and 48h samples and save them for later use. Use the method in Example 1 to extract RNA from different treatments and at different time points, and synthesize cDNA. Design Real-time PCR primers P3 and P4.
[0046]P3: 5'-GCTGAGGAGACTACAAAGACTG-3' (SEQ ID NO.5),
[0047] P4: 5'-ACATACCTCCAAGGCTTCCAAG-3' (SEQ ID NO.6),
[0048] according to Premix Ex TaqTM (Tli RNaseH Plus) (Dalian Bao Biology) kit prepared the reaction system, using ABI 7500thermocycler (Applied Biosystems, ...
Embodiment 3
[0052] Example 3 Construction of gene silencing vector and analysis of disease resistance of GhSNAP33 gene silencing plants
[0053] Design cotton gene silencing vector primers P7 and P8.
[0054] P7: 5'-CGACGACAAGACCGTGACCATGACTGTCAATAACTGTGTG-3' (SEQ ID NO.9),
[0055] P8: 5'-GAGGAGAAGAGCCGTCATTAGAGTAATCGACGAGCACGTTG-3' (SEQ ID NO.10),
[0056] Using the cDNA in Example 1 as a template, the gene fragment required for silencing was amplified. The target fragment was connected to the pTRV2 vector by vector fusion technology. The clone with the correct sequence was selected and transformed into Agrobacterium GV3010. According to the method of virus-induced gene silencing (Gao X, Wheeler T, Li Z, Kenerley C M, He P, Shan L, 2011, Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant J.66, 293-305.) Cotton with down-regulated expression of GhSNAP33 plant growth ( Figure 3A ). Cotton GhUBQ was used as a reference gene to identify the efficien...
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