茶树CsSMT基因的克隆、原核表达及植物超表达载体构建  

Cloning and Prokaryotic Expression of Selenocysteine Methyltransferase Gene from Tea Plant and Its Plant Overexpression Vector Construction

作　　者：刘声传[1] 鄢东海[1] 周雪[1] 曹雨[1] 莫雪[1] 胡伊然[1] 周玉锋[1] 

机构地区：[1]贵州省农业科学院茶叶研究所,贵州贵阳550006

出　　处：《西南农业学报》2016年第7期1540-1546,共7页Southwest China Journal of Agricultural Sciences

基　　金：贵州省农业科学院研究生科研创新基金项目"茶树富硒作用关键酶基因CsSMT的克隆与超表达研究"[黔农科合(创新基金)2011006];贵州省体改项目"现代高效农业园区茶叶栽培与加工技术集成示范"[黔科合Z字[2013]4008];贵州省农业攻关项目"贵定鸟王茶种质资源的保护及开发利用"[黔科合NY字[2011]3046];贵州省农业动植物育种专项"黔茶1号区域适应性高效栽培研究与示范"[黔农育专字(2012)022号];"贵定鸟王种扩繁和栽培技术集成与示范"[黔农育专字(2015)003]

摘　　要：为了发掘利用茶树(Camellia sinensis)富硒关键酶硒代半胱氨酸甲基转移酶(Selenocysteine methyltransferase,SMT)基因Cs SMT,基于宁州2号转录组测序结果,通过RACE克隆该基因的c DNA全长,并通过原核表达和蛋白质印迹(Western blotting)验证该基因,然后构建超表达载体,转化至根癌农杆菌。结果表明:该基因c DNA全长为1277 bp,开放阅读框(Open reading frame,ORF)为1 050 bp,与NCBI公布的茶树该基因ORF序列有8个点突变,且缺少GTCGTC序列。Cs SMT基因编码349个氨基酸,其氨基酸序列与毛果杨、野生大豆、黄芪的SMT以及川桑的同型半胱氨酸-S-甲基转移酶2(Homocysteine-S-methyltransferase 2,HMT2)的氨基酸序列有较高的相似性。目标蛋白分子量约38 k Da,为水溶性蛋白,与预测结果相符。成功构建Cs SMT基因的超表达载体,并获得携带Cs SMT的农杆菌菌株。To explore the selenecysteine methyltransferase （SMT） gene CsSMT, which plays an essential role in Camellia sinenzis leaves enriching selenium, based on transcriptome sequencing results of Camellia sinesis cv. ＇ Ningzhou 2＇ , the whole cDNA was obtained by RACE cloning. The target gene was identified by prokaryotlc expression and western blotting, integrated into overexpression vector, and then transformed to Agrebacterium tumefaciens. The results showed that the whole cDNA contained an open reading frame （ORF） of 1050 bp. In comparison to CsSMT ORF published in NCBI, it had eight point mutations and missed GTCGTC. In the present study, CsSMT encoded 349 amino acids, and shared high similarity with SMT from Populus trichocarpa, Clycine soja, and Astragalgs bisulcatus as well as homocysteine- S-methyltransfurase 2 （HMT2） in Morus notabilis. CsSMT was a water-soluble protein and its molecular weight was approximately 38 kDa, which was consistent with the putative results. The authors successfully constructed the overexpression vector of CsSMT and gained a strain of A. tumefaciens with the target gene.

关 键 词：茶树 硒 CsSMT 原核表达 超表达 

分 类 号：S571.1[农业科学—茶叶生产加工]