【摘要】：在實驗室前期構(gòu)建cDNA幼根文庫獲得谷氨酰胺合成酶(glutamine synthetase,GS,EC 6.3.1.2)同源序列(contig48)的基礎(chǔ)上設(shè)計引物,通過SMART RACE技術(shù)克隆了該基因cDNA全長序列(命名為GS1-2,GenBank登錄號:JQ925873.1)。結(jié)果顯示:(1)GS1-2基因全長為1 710bp,開放閱讀框長1 071bp,編碼356個氨基酸,預(yù)測蛋白分子質(zhì)量為39.3kD,理論等電點為5.65;核酸序列分析表明,GS1-2基因與從安吉白茶中克隆的茶氨酸合成酶基因相似性為99%。(2)將GS1-2基因克隆至原核表達(dá)載體pET-32a和pMAL-c5x,轉(zhuǎn)化至大腸桿菌,IPTG誘導(dǎo)表達(dá)融合蛋白,SDS-PAGE檢測結(jié)果表明,pET-32a-CsGS1-2轉(zhuǎn)至Rosetta中誘導(dǎo)表達(dá)的蛋白與預(yù)測蛋白大小一致,主要以包涵體形式存在;而pMAL-c5x-CsGS1-2轉(zhuǎn)化大腸桿菌BL21(DE3)誘導(dǎo)表達(dá)可產(chǎn)生可溶性蛋白。(3)進(jìn)一步構(gòu)建茶樹GS1-2酵母表達(dá)載體pYES-DEST52-CsGS1-2并轉(zhuǎn)化至釀酒酵母(WAT11)菌液中,添加底物(谷氨酸鈉100μmol/L和鹽酸乙胺500μmol/L)震蕩培養(yǎng)并離心,UPLC-MS測定酶反應(yīng)產(chǎn)物結(jié)果初步表明,目的蛋白不能催化鹽酸乙胺和谷氨酸鈉合成茶氨酸,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.
【作者單位】： 安徽農(nóng)業(yè)大學(xué)茶樹生物學(xué)與資源利用國家重點實驗室;
【基金】：國家自然科學(xué)基金(31170283,31300576)
【分類號】：Q943.2;S571.1

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