Promoter BgIosP556, and preparation method and application thereof
A technology of promoters and uses, applied in the field of promoters, can solve problems such as limited regulatory effects
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Embodiment 1P556
[0060] The PCR amplification of embodiment 1P556 promoter fragment and the construction of pMD18-T+P556 recombinant vector
[0061] PCR amplification
[0062] Use plant genome DNA extraction kit (TIANGEN new plant genome DNA extraction kit, catalog number: DP320-02) to extract rice Nipponbare seeds (preserved in Wuhan University Preservation Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province on December 18, 2009, That is, the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC No: 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 , add restriction enzyme site EcoR I and protection base, downstream primer R1, add restriction enzyme site Sbf I and protection base). Using the rice Nipponbare gDNA extracted above as a template, high-fidelity Ex Taq TM (TaKaRa, DRR100B) polymerase for PCR amplification. As shown in Table 1.
[0063...
Embodiment 2p8
[0083] Construction of embodiment 2p8+P556 recombinant vector
[0084] According to the operating manual of the TIANGEN common plasmid small extraction kit (catalogue number: DP103-03), the cloning vector with the P556 promoter sequence of the present invention is extracted from the Escherichia coli DH5α-P556 transformed with the promoter P556 constructed in Example 1 pMD18-T+P556; After purification, digest with the corresponding restriction enzymes EcoR 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.
[0085] Transform the resulting ligation product p8+P556 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, ...
Embodiment 3
[0111] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P556 cells
[0112] The p8+P556 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 tumefaciens EHA105 (preserved on December 24, 2009 in the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, namely the China Type Culture Collection Center (CCTCC), the preservation number is CCTCC No: M 209315) State cells, the specific method is as follows:
[0113] 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+P556 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen...
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