茶树谷氨酰胺合成酶同源基因的克隆及表达分析

摘要

In this study,based the homologous sequence of glutamine synthetase (contig48)screened out from young root cDNA library,we designed primers and cloned its full length (named as GS1-2,GenBank accession number:JQ925873.1)using SMART RACE.The results indicated that:(1)the full-length cD-NA of GS1-2 is 1710 bp with an open reading frame (ORF)of 1071 bp,encoding 356 amino acids with deduced molecular weight of 39 .3 kD and theoretical pI value of 5 .65 .The result of amino acid sequence a-lignment in NCBI indicated that it has high similarity with the cloned theanine synthetase from Anj i white tea.(2)The gene was cloned into prokaryotic expression vector pET-32a and pMAL-c5x,which was fur-ther transformed into Escherichiacoli Rosetta and BL21 to induce fusion protein with IPTG,respectively. The SDS-PAGE results showed that:after induction with IPTG,an insoluble inclusion body would be pro-duced in E.coli Rosetta using the pET-32a expression vector,while a soluble protein could be induced in E.coli BL21 (DE3 )using the pMAL-c5 x expression vector.(3 )After the yeast expression vector pYES-DEST52-CsGS1-2 was constructed by Gateway technology,the gene was expressed in Saccharomycescere-visiae (WAT11),and with addition of substrates in medium,the glutamine concentration in strains with pYES-GS1-2 vector transformant was twice as high as that containing the pYES empty vector transfor-mant using UPLC-MS analysis.The preliminary result suggested that the GS1-2 is capable of synthesizing glutamine instead of theanine.%在实验室前期构建cDNA幼根文库获得谷氨酰胺合成酶(glutamine synthetase,GS,EC 6.3.1.2)同源序列(contig48)的基础上设计引物,通过SMART RACE技术克隆了该基因cDNA全长序列(命名为GS1-2,GenBank登录号:JQ925873.1).结果显示:(1)GS1-2基因全长为1710 bp,开放阅读框长1071 bp,编码356个氨基酸,预测蛋白分子质量为39.3 kD,理论等电点为5.65;核酸序列分析表明,GS1-2基因与从安吉白茶中克隆的茶氨酸合成酶基因相似性为99%.(2)将GS1-2基因克隆至原核表达载体pET-32a和pMAL-c5x,转化至大肠杆菌,IPTG诱导表达融合蛋白,SDS-PAGE检测结果表明,pET-32a-CsGS1-2转至Rosetta中诱导表达的蛋白与预测蛋白大小一致,主要以包涵体形式存在;而pMAL-c5 x-CsGS1-2转化大肠杆菌BL21(DE3)诱导表达可产生可溶性蛋白.(3)进一步构建茶树GS1-2酵母表达载体pYES-DEST52-CsGS1-2并转化至酿酒酵母(WAT11)菌液中,添加底物(谷氨酸钠100μmol/L和盐酸乙胺500μmol/L)震荡培养并离心,UPLC-MS测定酶反应产物结果初步表明,目的蛋白不能催化盐酸乙胺和谷氨酸钠合成茶氨酸,但可以合成谷氨酰胺.