Promoter BgIosP582 and preparation method and application thereof
A technology of EHA105-P582 and promoters, which is applied in the field of promoters and can solve problems such as limited regulatory effects
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Embodiment 1P582
[0059] The PCR amplification of embodiment 1P582 promoter fragment and the construction of pMD18-T+P582 recombinant vector
[0060] PCR amplification
[0061] Using a plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02) to extract rice Nipponbare (preserved in Wuhan University Preservation Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province on December 18, 2009, namely China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC-P200910) Genomic DNA, according to the sequence of the promoter in the rice Nipponbare gDNA, a pair of PCR-specific amplification primers (upstream primer F1, upstream primer F1, Add restriction enzyme cutting site Kpn I and protective base, downstream primer R1, add restriction enzyme cutting site Sbf I and protective base). Using the rice Nipponbare gDNA extracted above as a template, high-fidelity Ex Taq TM (TaKaRa, DRR100B) polymerase for PCR amplification. As sh...
Embodiment 2p8
[0082] Construction of embodiment 2p8+P582 recombinant vector
[0083] According to the operating manual of the TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), extract the cloning vector with the P582 promoter sequence of the present invention from the Escherichia coli DH5α-P582 transformed with the promoter P582 constructed in Example 1 pMD18-T+P582; After purification, digest with the corresponding restriction enzymes Kpn I (NEB) and Sbf I (NEB), recover the corresponding promoter insert, and use the same restriction endonucleases as the p8 plasmid The large vector fragments recovered after digestion with Dicer enzyme were ligated.
[0084] Transform the resulting ligation product p8+P582 recombinant vector into competent cells 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, sing...
Embodiment 3
[0110] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P582 cells
[0111] The p8+P582 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). Competent cells of Agrobacterium EHA105 (preserved in China Center for Type Culture Collection (CCTCC) on December 24, 2009, with a preservation number of CCTCC M 209315), the specific method is as follows:
[0112] 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+P582 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen for 5 minutes, thaw at 37°C for 5 minutes, add 800 μl of normal temperature LB Liquid culture medium, reco...
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