茶树谷氨酰胺合成酶同源基因的克隆及表达分析
发布时间：2018-11-28 13:57
【摘要】：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。
[Abstract]:Primer was designed on the basis of constructing cDNA young root library to obtain the homologous sequence of glutamine synthase (glutamine synthetase,GS,EC 6.3.1.2) (contig48). The full-length cDNA sequence of the gene was cloned by SMART RACE (named GS1-2,GenBank accession number: JQ925873.1). The results showed that: (1) the total length of GS1-2 gene was 1 710 BP, the length of open reading frame was 10 71 BP, the predicted molecular weight of protein was 39. 3 KD and the theoretical isoelectric point was 5. 65; Nucleic acid sequence analysis showed that the similarity between GS1-2 gene and theanine synthase gene cloned from Anji White Tea was 99g. (2) the GS1-2 gene was cloned into prokaryotic expression vector pET-32a and pMAL-c5x, and transformed into Escherichia coli. IPTG induced the expression of fusion protein. The results of SDS-PAGE analysis showed that the expression of protein induced by pET-32a-CsGS1-2 in Rosetta was the same as that of predicted protein, mainly in the form of inclusion body. The expression of soluble protein was induced by pMAL-c5x-CsGS1-2 transformation into Escherichia coli BL21 (DE3). (3) the expression vector pYES-DEST52-CsGS1-2 of tea GS1-2 yeast was further constructed and transformed into the yeast solution of WAT11 (Saccharomyces cerevisiae). The substrates (sodium glutamate 100 渭 mol/L and ethylamine hydrochloride 500 渭 mol/L) were cultured and centrifuged. The results of UPLC-MS analysis showed that the target protein could not catalyze the synthesis of theanine hydrochloride and sodium glutamate. But glutamine can be synthesized.
【作者单位】： 安徽农业大学茶树生物学与资源利用国家重点实验室;
【基金】：国家自然科学基金(31170283,31300576)
【分类号】：Q943.2;S571.1
[Abstract]:Primer was designed on the basis of constructing cDNA young root library to obtain the homologous sequence of glutamine synthase (glutamine synthetase,GS,EC 6.3.1.2) (contig48). The full-length cDNA sequence of the gene was cloned by SMART RACE (named GS1-2,GenBank accession number: JQ925873.1). The results showed that: (1) the total length of GS1-2 gene was 1 710 BP, the length of open reading frame was 10 71 BP, the predicted molecular weight of protein was 39. 3 KD and the theoretical isoelectric point was 5. 65; Nucleic acid sequence analysis showed that the similarity between GS1-2 gene and theanine synthase gene cloned from Anji White Tea was 99g. (2) the GS1-2 gene was cloned into prokaryotic expression vector pET-32a and pMAL-c5x, and transformed into Escherichia coli. IPTG induced the expression of fusion protein. The results of SDS-PAGE analysis showed that the expression of protein induced by pET-32a-CsGS1-2 in Rosetta was the same as that of predicted protein, mainly in the form of inclusion body. The expression of soluble protein was induced by pMAL-c5x-CsGS1-2 transformation into Escherichia coli BL21 (DE3). (3) the expression vector pYES-DEST52-CsGS1-2 of tea GS1-2 yeast was further constructed and transformed into the yeast solution of WAT11 (Saccharomyces cerevisiae). The substrates (sodium glutamate 100 渭 mol/L and ethylamine hydrochloride 500 渭 mol/L) were cultured and centrifuged. The results of UPLC-MS analysis showed that the target protein could not catalyze the synthesis of theanine hydrochloride and sodium glutamate. But glutamine can be synthesized.
【作者单位】： 安徽农业大学茶树生物学与资源利用国家重点实验室;
【基金】：国家自然科学基金(31170283,31300576)
【分类号】：Q943.2;S571.1
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