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根据GenBank上与周期蛋白依赖性蛋白激酶基因相关序列,从苹果MdCKS基因编码区设计带有XbaⅠ和SalⅠ酶切位点的引物,以富士MdCKS基因的cDNA为模板将PCR扩增片段连接到pMD18-T Sim-ple Vector上,测序正确后,再连接到植物表达载体pBI121上,经过抗性筛选和测序验证,成功构建了MdCKS基因表达载体。并通过电击转化法导入农杆菌LBA4404,以备将来用于番茄转化。
According to the sequence related to cyclin-dependent protein kinase gene in GenBank, primers with XbaⅠand SalⅠ restriction sites were designed from the coding region of MdCKS gene of apple and the cDNA fragment of Fuji MdCKS gene was used as a template to ligate the PCR fragment into pMD18- T Sim-ple Vector. After sequencing correctly, it was ligated to the plant expression vector pBI121. The MdCKS gene expression vector was successfully constructed after resistance screening and sequencing verification. Agrobacterium tumefaciens LBA4404 was transformed by electroporation to prepare for future tomato transformation.