紫杆柽柳谷胱甘肽硫转移酶基因的克隆及功能鉴定  

Cloning and Function Characterization of Glutathione S-transferase Gene from Tamarix androssowii

作　　者：杨平[1] 胡军[1] 王玉成[2] 尹维波[1] 陈宇红[1] 姜静[2] 李集临[3] 胡赞民[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所,北京100101 [2]东北林业大学林学院,哈尔滨150040 [3]哈尔滨师范大学生命与环境科学学院,哈尔滨150086

出　　处：《农业生物技术学报》2007年第1期76-80,共5页Journal of Agricultural Biotechnology

基　　金：国家转基因植物研究与产业化开发专项(No.JY03A1802)资助。

摘　　要：根据GenBank已公布的植物谷胱甘肽硫转移酶基因EST序列设计引物,利用3'和5'RACE方法,获得紫杆柽柳GST基因(TaGST)全长cDNA序列,全长为1175bp,开放读码区672bp,编码224个氨基酸(GenBank accission No.DQ683363)。其所编码的蛋白与大豆的GST10同源性为48%。利用pET-28a(+)构建了紫杆柽柳(Tamarix androssowii)GST基因的原核表达载体,转入大肠杆菌(Escherichia coli)BL21进行了原核表达。诱导表达蛋白的SDS-PAGE检测表明,所表达的蛋白分子量约为26kD。初步的酶学活性检测表明,所克隆的基因编码的肽段具有催化GST蛋白通用底物CDNB(1-氯-2,4-二硝基苯)和GSH(还原型谷胱甘肽)反应的活性。Based on the EST sequence of glutathione S-transferase gene from Tamarix androssowii （TaGST） published on GenBank, a TaGSTcDNA was cloned with 3＇ and 5＇ RACE techniques. The full length of TaGSTwas 1 175 bp with ORF of 672 bp that encoded 224 amino acid residues（GenBank accission No. DQ683363）. The deduced amino acid sequence encoded by TaGST was homologous with that of known plant GST and the identity was 48% （GST10 of Glycine max ）. To characterize its function, the TaGST expression vector was constructed with pET-28a （＋） and transformed into Escherichia coli BL21. The SDS-PAGE result showed that the TaGST protein was expressed and the molecular weight was about 26 kD which was similar to the expected one. The enzyme activity assay of the TaGST indicated that TaGST could catalyze the substrate reaction of CDNB（1-chloro-2,4-dinitrobenzene） with GSH （reduced glutathione）. It was confirmed that the TaGSTdid encode a functional GST protein.

关 键 词：紫杆柽柳 谷胱甘肽硫转移酶 基因克隆 原核表达 酶活性检测 

分 类 号：S188[农业科学—农业基础科学]