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在TMV外壳蛋白(CP)基因的5′和3′端分别合成寡聚核苷酸引物P1和P2,并分别引入ClaI和BamHI位点,采用PCR技术扩增TMVCP基因,用ClaIBamHI消化后重组到pBluescriptKS+中,并将该基因与CMVCP基因重组在同一个表达载体pE3上获得双价CP基因表达载体pETC2,转化番茄,获得再生植株。通过点杂交、PCR检测和Southern杂交,证实2、12和13号为转TMV和CMVCP基因的工程植株。工程植株在温室中表现正常,比对照植株表现出明显的抗性。
The oligonucleotide primers P1 and P2 were synthesized at the 5 ’and 3’ ends of the TMV coat protein (CP) gene and introduced into ClaI and BamHI sites respectively. The TMVCP gene was amplified by PCR and digested with ClaIBamHI Recombinant pBlue scrips +, and the gene and CMVCP gene recombination in the same expression vector pE3 obtained bivalent CP gene expression vector pETC2, transformed tomato, get regenerated plants. Through dot blot, PCR and Southern hybridization, it was confirmed that the transgenic plants 2, 12 and 13 were TMV and CMVCP genes. The engineered plants behaved normally in the greenhouse and showed significantly greater resistance than the control plants.