A promoter sbubi1, its preparation method and use
A kind of technology of promoter and purpose, applied in the field of promoters of plants such as sweet sorghum, and can solve problems such as large expression amount
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Embodiment 1
[0060] Example 1: PCR amplification of the P604 promoter fragment and pMD18-T+P604 recombination body construction
[0061] PCR amplification
[0062] Use plant genomic DNA extraction kit (TIANGEN novel plant genomic DNA extraction kit, catalog number: DP320-02) to extract the genomic DNA of sweet sorghum BT×623 seeds, according to the sequence of this promoter in sweet sorghum BT×623gDNA, respectively Design a pair of PCR-specific amplification primers at the beginning and end (upstream primer F1, plus restriction enzyme site KpnI and protective base, downstream primer R1, plus restriction enzyme site PstI and protective base). Using the above extracted gDNA of sweet sorghum BT×623 as a template, high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase was used for PCR amplification. As shown in Table 1.
[0063] Table 1 PCR system for gene promoter amplification
[0064]
[0065] The PCR amplification program was as follows: 94°C pre-denaturation for 5 min, then denatur...
Embodiment 2
[0082] Embodiment 2: Construction of carrier-p8+P604 recombinant vector
[0083] According to the operating manual of the TIANGEN ordinary plasmid small extraction kit (catalogue number: DP103-03), the cloning vector with the P604 promoter sequence of the present invention is extracted from the Escherichia coli DH5α-P604 transformed with the promoter P604 constructed in Example 1 pMD18-T+P604; After purification, digest with the corresponding restriction enzymes Kpn I (NEB) and Pst I (NEB), recover the corresponding promoter insert fragment, and use the same restriction endonuclease as p8 plasmid The large vector fragments recovered after digestion with Dicer enzyme were ligated.
[0084] Transform the resulting ligation product p8+P604 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, and the tr...
Embodiment 3
[0110] Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P604 cells
[0111] The p8+P604 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 on December 24, 2009 at the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, that is, the China Type Culture Collection Center (CCTCC), the preservation number is CCTCC M 209315), and the specific method 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+P604 recombinant vector and p8 plasmid and p8 empty vector as a control, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen for...
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