Promoter BgIosP535, and preparation method and use thereof
A technology of promoters and nucleic acid constructs, applied in botany equipment and methods, biochemical equipment and methods, horticultural methods, etc., can solve problems such as limited regulatory effects
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Embodiment 1
[0080] Example 1: PCR amplification of P535 promoter fragment and construction of pMD18-T+P535 recombinant vector PCR amplification
[0081]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-specific amplification was designed at the beginning and the end respectively. Primers (upstream primer F1, plus restriction site KpnI and protective bases, downstream primer R1, plus restriction site SbfI and protective bases). 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.
[0082] Table 1 PCR system for gene promoter amplification
[0083]
[0084] The PCR ampl...
Embodiment 2
[0099] Embodiment 2: Construction of carrier-p8+P535 recombinant vector
[0100] According to the operating manual of the TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), extract the cloning vector with the P535 promoter sequence of the present invention from the Escherichia coli DH5α-P535 transformed with the promoter P535 constructed in Example 1 pMD18-T+P535; 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.
[0101] The resulting ligation product p8+P535 recombinant vector was transformed into competent cells DH5α prepared according to the calcium chloride method shown in "Molecular Cloning Experiment Guide" (third edition, Science Press), and cultured upside down at 37°C for 16-24 hours. After the transformants grow into colonies, single clon...
Embodiment 3
[0127] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P535 cells
[0128] The p8+P535 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were respectively transformed into root cancer prepared by the calcium chloride method described in the "Molecular Cloning Experiment Guide" (third edition, Science Press). Agrobacterium EHA105 (preserved in the invention application with the application number 200910238992.0 and the invention title "A promoter BgIosP587, its preparation method and use", and published on September 22, 2010, with the preservation number CCTCC M 209315) Competent cells, the specific method is as follows:
[0129] 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+P535 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid ni...
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