烟草CYP71基因的克隆与原核优化表达  

Cloning and Optimized Prokaryotic Expression of Tobacco CYP71 Gene

作　　者：佟硕秋 高洁[1] 钟杰[1] 贡献 吴拥军[1] TONG Shuo-qiu;GAO Jie;ZHONG Jie;GONG Xian;WU Yong-jun(College of Life Sciences,Guizhou University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学生命科学学院,贵州贵阳550025

出　　处：《山地农业生物学报》2018年第1期12-16,共5页Journal of Mountain Agriculture and Biology

基　　金：国家自然科学基金项目(No.31071755/C1408)

摘　　要：本文从烟草中克隆出CYP71基因,进行生物信息学分析,并在原核细胞中表达出目的蛋白。应用RTPCR,从野生型烟草(Nicotiana tabacum)叶片中克隆出一种CYP450 c DNA序列(CYP71)。序列分析表明,烟草CYP71基因ORF长为1545 bp,编码514个氨基酸,基因登录号为KC480444.1。将去除跨膜信号肽及密码子优化后的基因合成后,构建至p ET-30a表达载体,转化到大肠杆菌Rosetta(DE3)中,经IPTG诱导和SDS-PAGE电泳、Western-blot检测,获得与预期一致的49 KDa蛋白质,为进一步研究CYP71基因的抗虫功能奠定了基础。The aim of the present study was to obtain CYP71 gene from tobacco to conduct bioinformatic analysis,as well as to obtain the expressed protein in prokaryotic cells.The cDNA sequence of CYP71(GenBank accession number:KC480444.1)was obtained using RT-PCR method.Its open reading frame(ORF)was 1,545 bp in length,encoding 514 amino acids.The coding sequence of the CYP71 gene was optimized by modification of codon usage and removal of transmembrane signal peptide.The recombinant plasmid pET-30a was transformed into E.coli Rosetta(DE3).The recombinant protein with predicted molecular weight was successfully induced to express by IPTG and was detected and confirmed by SDS-PAGE and Western blot.The results will lay a foundation for further study and utilization of its insect resistance function.

关 键 词：烟草 CYP71 基因克隆 生物信息学分析 原核表达 

分 类 号：Q78[生物学—分子生物学]