Cotton TRY gene
A cotton, gene technology, applied in genetic engineering, plant genetic improvement, angiosperms/flowering plants, etc.
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Embodiment 1
[0034] Example 1 Cloning of Cotton TRY Gene
[0035] 1. Cultivation of plant materials
[0036] The cotton Xuzhou 142 lint-free and floc-free mutant seeds after delinting were soaked in tap water for imbibition and placed in a 28°C incubator for 48 hours to accelerate germination. The cotton seeds that germinated in white were planted in flower pots and cultivated in a light culture room for more than 20 days , you can grow 3-5 true leaves. Fresh young cotton leaves were used for total RNA extraction.
[0037] 2. Extraction of cotton total RNA
[0038] The general plant RNA extraction kit (spin column type) produced by Beijing Biotec Biotechnology Co., Ltd. was used to extract total RNA from young cotton leaves. The extraction process was carried out in strict accordance with the instructions, and the total RNA was stored at -70°C after extraction. Aspirate 3 μL of total RNA, and check the integrity of RNA by 1% agarose gel electrophoresis. Trace DNA in total RNA was remov...
Embodiment 2
[0064] Example 2 Construction of pBI121-TRY plant expression vector
[0065] see figure 2 , the TRY gene fragment and the expression vector pBI121 were digested with XbaI and BamHI endonucleases, and the excised TRY gene was inserted between the 35S promoter and the NOS terminator of the pBI121 expression vector to construct the plant expression vector pBI121-TRY .
[0066] Double Enzyme Cut Cotton TRY Gene Fragment
[0067] The cotton cDNA library was amplified by PCR with primers DR-1 and DR-2 designed with XbaI and BamHI restriction sites, and the amplified product was recovered to obtain a TRY gene fragment with XbaI and BamHI restriction sites. The recovered gene fragment was then double-enzyme-digested with XbaI and BamHI. See Table 4 for the enzyme-digestion system.
[0068] The primer sequences are as follows:
[0069] Upstream primer DR-1: 5′-GC TCTAGA ATGGACAAACGTGACCG-3';
[0070] Downstream primer DR-2: 5′-CG GGATCC TCAAACAGAACCGTGTCTC-3′;
[0071] The u...
Embodiment 3
[0074] Embodiment 3 Utilizes the transformation method mediated by Agrobacterium to obtain transgenic TRY tobacco
[0075] 1. Preparation of Agrobacterium Competent Cells
[0076] Pick a single colony of EHA105 and inoculate it in 5mL of YEB liquid medium (containing streptomycin 100mg / mL, rifampicin 100mg / mL), culture overnight at 28°C, 250rpm; transfer 2mL of the culture into 50mL of YEB liquid medium, and continue to cultivate until the OD600 value is 0.6; transfer the bacterial solution to a sterile centrifuge tube, ice-bath for 30 minutes, and centrifuge at 5000 rpm for 5 minutes; Aliquot 200 μL of the tube into sterile 1.5mL microcentrifuge tubes and keep at -70°C for use.
[0077] 2. Freeze-thaw method to transfer recombinant plasmid pBI121-CRY into Agrobacterium
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