酿酒酵母海藻糖合成酶基因的克隆和在大肠杆菌中的表达  

Cloning and Expression of TPS 1 Gene in Escherichia coli

作　　者：杨波[1] 戴秀玉[1] 周坚[1] 

机构地区：[1]中国科学院微生物研究所,北京100080

出　　处：《Acta Genetica Sinica》2001年第4期372-378,共7页

基　　金：国家自然科学基金资助项目!(批准号:39980023)

摘　　要：用ＰＣＲ方法克隆了１．５ｋｂ的酿酒酵母Ｓａｃｃｈｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ海藻糖合成酶基因ＴＰＳ１，将该片段连接到ｐＵＣ１９载体，通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　ＦＦ４１６９和ＦＦ４１６９和ＦＦ４０５０。对转化株的质粒ＤＮＡ酶切分析表明均含有１．５ｋｂＰＣＲ克隆片段。生长曲线实难证明，带有克隆片段的转化株在含０．５ｍｏｌ／ＬＮａＣｌ的高渗透压基础培养基中生长良好；用高效液相色谱（ＨＰＬＣ）结合蒸发光散射（ＥＬＳＤ）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。A S. cerevisiae TPS 1 gene for trehalose-6-phosphate synthase was cloned by PCR amplification. The 1.5kb DNA fragment was ligated to pUC19 and transformed into otsA deficient and deleted of E. coli strains FF4169 and FF4050 se parately. otsA gene is encoding trehalose-6-phasphate synthase in E.coli. Restriction endonucleases digestion analysis of transformants' plasmid DNA showed that there was a cloned 1.5kb fragment carried on the vector. The growth curve e xperiment result showed that the both transformants could grow well as wild type. Trehalose was synthesized and accumu lated in these transformants during high osmotic stress by HPLC conbined with ELSD (Evaporate light scatter detactor) determination. From the result above, we could conclude that the TPS 1 gene of S.cerevisiae was able to restore otsA g ene function in E.coli for both osmotolerance and trehalose accumulation during salt stress.

关 键 词：TPS1基因 海藻糖 PCR克隆 表达 酿酒酵母 海藻糖合成酶 

分 类 号：Q319[生物学—遗传学]