Construction of tomato expression vectors of EFE gene
2008
Mi Guoqiao | Li Jigang | Jia Yongliang
Chinese. 为研究蚯蚓纤溶酶的番茄表达，构建EFE基因的番茄表达载体pF和pEF，并导入农杆菌。采用PCR法在EFE基因DNA序列上添加了表达调控元件和酶切位点后，得到新EFE基因片段，将该片段与切去GUS基因的pBI121载体相连，以构建CaMV35S驱动的EFE组成型表达载体pF；用PCR克隆得到的E8启动子片段替换pF上的35S启动子，以构建番茄成熟果实特异性表达载体pEF；最后，采用三亲交配法用重组质粒转化根癌农杆菌EHA105；结果表明：经过酶切、PCR和测序鉴定，EFE基因、E8启动子序列已重组到表达载体上，植物表达载体构建正确，并且已经转化进农杆菌EHA105中。[著者文摘]
Show more [+] Less [-]English. The study aims to construct tomato EFE expression vectors pF and pEF and introduce these vectors into Agrobacterium, which will lay foundation for EFE expression in tomato plant. Expression regulation element and restriction sites were added to the EFE gene by PCR. The modi- fied EFE sequence was fused with vector pBI121 with GUS ORF being deleted. Thus, EFE was placed to the downstream of CaMV35S promoter in the binary vector pBI121, forming the recombi- nant plasmid pF; PCR- amplified E8 sequence was used to replace 35S promoter, resulting in the recombinant plasmid pEF. Finally, the recombinant plasmids were introduced into Agrobacterium tumefaciens EHA105 by tri - parental conjugation. The results showed that EFE gene and promoter sequence of E8 gene were recombinanted to the expression vectors which were analyzed by restric- tion enzyme digestion, PCR amplification, and sequencing. Plant expression vectors were success- fully constructed and introduced into Agrobacterium tumefaciens EHA105.
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