Construction of Fusion Expression Vector of α-galactosidase-EGFP in Cucumber

黄瓜α-半乳糖苷酶基因-EGFP融合表达载体的构建(摘要)(英文)

[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.

[目的]构建由CaMV35S启动子调控的黄瓜α-半乳糖苷酶的增强型绿色荧光蛋白(enhanced green flurescent protein,EGFP)融合表达载体。[方法]运用聚合酶链式反应(polymerase chain reaction,PCR)技术,分别以质粒pCambia1303和pEGFP-N1为模板扩增CaMV35S启动子序列和EGFP编码序列;运用反转录聚合酶链式反应(Reverse transcript-PCR,RT-PCR)技术,以黄瓜总RNA为模板扩增黄瓜酸性α-半乳糖苷酶Ⅰ编码序列;将上述3个片段插入表达载体pCambia 1381*c的多克隆位点,构建EGFP位于目的基因C端的α-半乳糖苷酶基因的EGFP融合表达载体。[结果]经酶切、测序等验证,黄瓜α-半乳糖苷酶-EGFP融合表达载体构建成功。[结论]为进一步研究黄瓜α-半乳糖苷酶的亚细胞定位奠定实验基础。