BGIos1013 gene and application thereof
A technology for transgenic plants, applied in the field of genetics, can solve the problem of less fine positioning of grain weight genes
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Embodiment 1B
[0063] The PCR amplification of embodiment 1 BGIos1013 gene and the construction of pMD18-T+BGIos1013 recombinant vector
[0064] PCR amplification
[0065] Genomic DNA of common wild rice Yuanjiang (Oryza. Design a pair of PCR-specific amplification primers (upstream primer F1, plus restriction site BamHI and protective bases, downstream primer R1, plus restriction site XbaI and protective bases). Using the gDNA extracted from Yuanjiang as a template, high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase was used for PCR amplification. As shown in Table 1.
[0066] Table 1 PCR system for target gene amplification
[0067]
[0068] The PCR amplification program was as follows: 94°C pre-denaturation for 5 min, then denaturation at 94°C for 45 s, annealing at 55°C for 50 s, extension at 72°C for 90 s, 35 reaction cycles, and finally 72°C extension for 7 min.
[0069] Among them, the upstream primer F1: GC GGATCC ACAATGCACTCACTGAAAC (SEQ ID NO: 2), containing a BamHI restr...
Embodiment 2p6
[0081] The construction of embodiment 2p6 recombinant vector
[0082] 1) PCR amplification of maize Ubiquitin promoter fragment and construction of pMD18-T+Ubi recombinant vector
[0083] Ubi promoter PCR amplification
[0084] Genomic DNA of maize variety B73 (Zea mays mays cv.B73) was extracted using a plant genomic DNA extraction kit (TIANGEN New Plant Genomic DNA Extraction Kit, catalog number: DP320-02) (http: / / www.maizegdb.org / ), according to the sequence of the promoter in maize B73gDNA, a pair of PCR-specific amplification primers were designed at the beginning and the end respectively (upstream primer F2: GG CTGCAG TGCAGCGTGACCCGGTCGT (SEQ ID NO: 4), plus restriction site Pst I and protection base, downstream primer R2: GG CTGCAG AAGTAACACCAAAC (SEQ ID NO: 5), plus restriction site Pst I and protection bases). Using the above extracted gDNA of corn B73 as a template, high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase was used for PCR amplification. As shown in...
Embodiment 3
[0132] Construction of embodiment 3p6+BGIos1013 recombinant vector
[0133] Extract the pMD18-T+BGIos1013 plasmid, and recover the BGIos1013 gene fragment after digestion with the restriction endonuclease BamHI / XbaI, and extract the p6 plasmid at the same time, and recover the large fragment after digestion with the corresponding restriction endonuclease BamHI / XbaI. The recovered product was ligated, and then the competent cell DH5α was transformed to obtain the recombinant vector p6+BGIos1013. Positive clones were screened for PCR detection and then sequenced for confirmation.
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