烟草,CYP71,基因的克隆与原核优化表达
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 RT-PCR，从野生型烟草（Nicotiana tabacum）叶片中克隆出一种 CYP450 cDNA 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 KC4804441。将去除跨膜信号肽及密码子优化后的基因合成后，构建至 pET-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。
关键词：烟草； CYP71 ；基因克隆；生物信息学分析；原核表达
中图分类号：Q78文献标识码：A
文章编号：1008-0457（2018）01-0012-05国际DOI编码：10.15958/j.cnki.sdnyswxb.2018.01.003
Cloning and Optimized Prokaryotic Expression of Tobacco CYP71 Gene
TONG Shuoqiu，GAO Jie，ZHONG Jie，GONG Xian，WU Yongjun*
（College of Life Sciences， Guizhou University， Guiyang， Guizhou 550025， China）
Abstract： 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.
Key words：tobacco； CYP71； ge...
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