酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI，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FF4050，对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段，生长曲线实验证明，带有克隆片段的转化株在含0．5mol/L NaCl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。

Cloning and Expression ofTPS1 Gene in Escherichia coli

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.