BgIosP585 promoter, preparation method and application
A technology of promoters and uses, applied in the field of genetic engineering, can solve problems such as limited regulatory effect
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Embodiment 1
[0072] Example 1: PCR amplification of P585 promoter fragment and construction of pMD18-T+P585 recombinant vector
[0073] PCR amplification
[0074] Use the plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02) to extract rice Nipponbare (the application number is 200910238992.0, the invention name is "a kind of promoter BgIosP587, its preparation method and use) According to the sequence of the promoter in the rice Nipponbare gDNA, a pair of PCR specificity was designed at the beginning and end respectively. Amplification primer (upstream primer F1, plus restriction enzyme site EcoR I and protection base, downstream primer R1, plus restriction enzyme site SbfI and protection base). Using the gDNA of rice Nipponbare extracted above as a template, high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase was used for PCR amplification. As shown in Table 1.
[0075] Table 1 PCR system for gene promoter amplification
[0076] ...
Embodiment 2
[0118] Embodiment 2: Construction of carrier-p8+P585 recombinant vector
[0119] According to the operating manual of the TIANGEN common plasmid small extraction kit (catalogue number: DP103-03), the cloning vector with the P585 promoter sequence of the present invention is extracted from the Escherichia coli DH5α-P585 transformed with the promoter P585 constructed in Example 1 pMD18-T+P585; After purification, digest with the corresponding restriction endonucleases KpnI and SbfI, recover the corresponding promoter insert fragment, and use the same restriction enzymes as the p8 plasmid to recover the vector Large fragments are concatenated.
[0120] Transform the resulting ligation product p8+P585 recombinant vector into the competent cell DH5α prepared according to the calcium chloride method shown in the "Molecular Cloning Experiment Guide" (third edition, Science Press), and culture it upside down at 37°C for 16-24 hours. After the colonies grow out, single clones are pi...
Embodiment 3
[0150] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P585 cells
[0151] The p8+P585 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were respectively transformed into the root cancer prepared by the calcium chloride method described in the "Molecular Cloning Experiment Guide" (third edition, Science Press). Agrobacterium EHA105 (has been preserved in the invention application with the application number 200910238992.0 and the invention title "A promoter BgIosP587, its preparation method and use", and was published on September 22, 2010, and the preservation number is CCTCC NO: M 209315) competent cells, the specific method is as follows:
[0152] The Agrobacterium tumefaciens competent cells EHA105 were taken out from the ultra-low temperature refrigerator and thawed on ice. After thawing, add 5 μl of p8+P585 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minut...
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