茶树谷氨酰胺合成酶同源基因的克隆及表达分析

Clone and Expression of GS Homologue of Tea Plant(Camellia sinensis)

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1 710bp,开放阅读框长1 071bp,编码356个氨基酸,预测蛋白分子质量为39.3kD,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-c5x-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。

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 cDNA of GS1 2 is 1 710 bp with an open reading frame （ORF） of 1 071 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 alignment in NCBI indicated that it has high similarity with the cloned theanine synthetase from Anji white tea. （2） The gene was cloned into prokaryotic expression vector pET 32a and pMAL c5x, which was further transformed into Escherichia coli 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 produced 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 c5x expression vector. （3） After the yeast expression vector pYES DEST52 CsGS1 2 was constructed by Gateway technology, the gene was expressed in Saccharomyces cerevisiae （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 transformant using UPLC MS analysis. The preliminary result suggested that the GS1 2 is capable of synthesizing glutamine instead of theanine.