Novel rice DREB transcription factor OsDREBx and coding gene and application thereof
A technology of transcription factor and rice, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as limited effect
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Embodiment 1
[0025] Example 1 Cloning of OsDREBx gene
[0026] In the present invention, the full-length sequence of the OsDREBx gene is cloned from the indica rice variety 9311 by the following method.
[0027] The total DNA of the indica rice variety 9311 was extracted using a genomic DNA purification kit (purchased from Cybersun Corporation). Design primer sequence as follows:
[0028] P1: 5’ATGGCACTGG GGTTATTGAA CGC 3’
[0029] P2: 5’TCACAAGC CCTCAAAGAA CTGAG 3
[0030] PCR reaction system:
[0031] 10×buffer 2.5μl
[0032] MgCl 2 (25mM) 1.5μl
[0033] Taq enzyme 0.2μl
[0034] dNTPs(2.5mM) 2μl
[0035] Primer 2μl
[0036] Template 5μl
[0037] Final reaction volume 25μl
[0038] Thermal cycle reaction: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing temperature is determined by primers, 72°C extension for 2 min, 30 cycles; 72°C extension for 5 min; 4°C to stop the reaction. The amplification reaction products were electrophoresed on a 1% agarose gel, and the PCR amplific...
Embodiment 2
[0039] Example 2 Verification of OsDREB gene in yeast one-hybrid binding experiment
[0040] The constructed yeast pGADT7-Rec2 vector containing the OsDREBx gene, and the constructed pHis-DRE4 vector containing four DRE (TACCGACTA) repeats, which can bind to both DREB1 and DREB2, were co-transformed into the Y187 competent strain and spread on On the triple deficiency medium SD / -His / -Leu / -Trp containing 100Mm 3AT, it grows like figure 2 The colony shown in A; At the same time, in the control, whether the yeast pGADT7-Rec2 vector without the OsDREBx gene and the pHis-DRE4 vector containing the four DRE repeats were co-transformed into the Y187 competent strain, or The constructed yeast pGADT7-Rec2 vector containing the OsDREBx gene was co-transformed into the Y187 competent strain with the pHis blank vector not linked to the four DRE repeats, and spread on the triple deficiency medium SD / -His / - containing 100Mm3AT After Leu / -Trp, no colonies grew.
[0041] In addition, in order t...
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