忽地笑CYP98A的分子克隆与表达分析  

Molecular Cloning and Expression Analysis of CYP98A from Lycoris aurea

作　　者：钱彬彬[1] 李宜奎[1] 李洁 程丽[1] 夏冰[1] 汪仁[1] Qian Binbin;Li Yikui;Li Jie;Cheng Li;Xia Bing;Wang Ren(Institute of Botany, Jiangsu Province and Chinese Academy of Seienees, Nanjing, 210014)

机构地区：[1]江苏省中国科学院植物研究所,南京210014

出　　处：《分子植物育种》2018年第11期3533-3541,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31600074);江苏省自然科学基金项目(BK20160599);江苏省植物资源研究与利用重点实验室基金项目(JSPKLB201505)共同资助

摘　　要：利用RACE技术从忽地笑(Lycoris aurea(L’Hér.)Herb.)花葶中克隆获得细胞色素P450酶98A亚家族蛋白编码基因LaCYP98A。该基因cDNA全长1 794 bp,开放阅读框为1 518 bp,编码含有505个氨基酸残基的LaCYP98A蛋白。LaCYP98A具有细胞色素P450酶特征性的亚铁血红素结合结构域和半胱氨酸残基组成的活性中心。LaCYP98A与拟南芥、大豆等其他植物CYP98A氨基酸序列的一致性在60%以上,且具有CYP450的所有特征模序。LaCYP98A定位于植物细胞的内质网,其N-端29个氨基酸对于其亚细胞定位具有重要的作用。利用pET表达系统,通过截掉其N-端内质网定位区域,可以实现LaCYP98A在大肠杆菌中的诱导表达。本研究为LaCYP98A的功能鉴定和植物细胞色素P450的原核表达及其催化的次级代谢产物的异源合成奠定了基础。In this study, a gene encoding cytochrome P450 98 A subfamily was cloned from the scape of Lycoris aurea（L＇Hér.） Herb., which was named as LaCYP98 A.The full-length cDNA of LaCYP98 A is 1794 bp with a1518 bp open reading frame, encoding a deduced polypeptide of 505 amino acids. LaCYP98 A protein contains cytochrome P450 typical active center domains consisting of heme-binding motif and cysteine. LaCYP98 A shares more than 60% identity with CYP98 A from Arabidopsis thaliana, Glycine max and other plants, and owns all the motifs of CYP450. LaCYP98 A protein was located in the endoplasmic reticulum, and the N-terminal 29 amino acids was of importance to the subcellular localization of LaCYP98 A. Based on the pET expression system,LaCYP98 A was successfully expressed in Escherichia coli via truncating N-terminal localization region. In this study, molecular cloning and expression analysis of Lycoris aurea CYP98 A were investigated to facilitate further research in functional characterization and prokaryotic expression of plant CYP450 as well as plant secondary metabolites biosynthesis in E. coli.

关 键 词：忽地笑 CYP98A 分子克隆 亚细胞定位 原核表达 

分 类 号：Q943.2[生物学—植物学] S567.219[农业科学—中草药栽培]