拟南芥海藻糖酶基因克隆及其在大肠杆菌中高效表达与功能研究  

Cloning of Trehalase Gene from Arabidopsis thaliana,Over-expression in E.coli and Its Functional Activity Assay in vitro

作　　者：郭蓓[1] 胡磊[2] 盖颖[1] 王喜智[1] 陈雪梅[1] 蒋湘宁[1] 

机构地区：[1]北京林业大学生物科学与技术学院国家林业局树木花卉育种生物工程重点开放实验室 [2]南昌大学中德联合研究院教育部重点实验室,南昌330047

出　　处：《微生物学通报》2008年第2期241-248,共8页Microbiology China

基　　金：国家自然科学基金(No.30571512);北京市高校人才强教计划(No.PXM2007-014207-044539)

摘　　要：从拟南芥幼苗中提取RNA,通过RT-PCR克隆得到海藻糖酶基因后,将其构建到原核高效表达载体pET30a(+)上并在大肠杆菌BL21菌株中进行高效诱导表达,继而对纯化得到的海藻糖酶蛋白进行活性检测和酶学特性研究。实验结果表明,植物源的海藻糖酶基因在异体大肠杆菌中能够高效表达,纯化获得的海藻糖酶蛋白在试管条件下具有较高的海藻糖水解活性,其活性最适温度为45℃。通过GC-MS分离检测,可以明显地看到酶反应过程中底物海藻糖和产物葡萄糖的含量随反应时间变化的消长关系,这充分证明克隆基因在大肠杆菌中的表达产物具有海藻糖酶的功能。The molecular researches of trehalose biosynthesis and metabolization have been attended because of its important roles in the cyto-physiology process of animal and plant cell. By using RT-PCR （reverse transcription PCR） method, the cDNA sequence of trehalase has been cloned through the RNA extracted from Arabidopsis sprout in this experiment. 1674 bp cDNA identified by sequencing was constructed to pET30a （＋） vector and transformed to BL21 of E. coli. The target protein has overexpressed and has been purificated by using Ni-NTA Agarose, and then its activity assay and enzymatic characteristic were studied in vitro. The results indicated that this plant trehalase gene can overexpress in E. coli and the trehalase protein which purificated through Ni-NTA Agarose has higher hydrolysis activity and its suitable reaction temperature is 45℃. The quantity change between glucose and trehalose occurred obviously in the enzymatic reaction as time through GC-MS analysis. This further confirmed that the trehalase gene come from Arabidopsis has expressed functionally in E. coli.

关 键 词：拟南芥 海藻糖酶 基因克隆 功能表达 

分 类 号：Q943.2[生物学—植物学]