BGIos228 gene and application thereof
A technology for use and transgenic plants, applied in the field of genes, can solve the problems of gene research related to rice differentiation and emergence that have not been seen
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Embodiment 1
[0056] Example 1: PCR amplification of BGIos228 gene and construction of pMD18-T+BGIos228 recombinant vector
[0057] PCR amplification
[0058] Genomic DNA (gDNA) of common wild rice Yuanjiang (Oryza. , respectively design a pair of PCR-specific amplification primers at the beginning and end (upstream primer F1, plus restriction site BamHI and protection base, downstream primer R1, plus restriction site Xba I and protection base). 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.
[0059] Table 1 PCR system for target gene amplification
[0060]
[0061] Composition Volume (μl)
[0062]
[0063] Genomic DNA 0.2
[0064] dNTPs (2.5mM) 2
[0065] 10×Ex Buffer (with magnesium ions) 2.5
[0066] Primer F1 (50μM) 1
[0067] Primer R1 (50μM) 1
[0068] Ex taq 0...
Embodiment 2
[0085] Embodiment 2: Construction of p6 recombinant vector
[0086] 1) PCR amplification of maize Ubiquitin promoter fragment and construction of pMD18-T+Ubi recombinant vector
[0087] Ubi promoter PCR amplification
[0088] 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 t...
Embodiment 3
[0154] Embodiment 3: Construction of p6+BGIos228 recombinant vector
[0155] Extract the pMD18-T+BGIos228 plasmid, and recover the BGIos228 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 product was ligated, and then the competent cell DH5α was transformed to obtain the recombinant vector p6+BGIos228. Positive clones were screened for PCR detection and then sequenced for confirmation.
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