副干酪乳杆菌6-磷酸山梨醇脱氢酶(gutF)基因的克隆和超表达

Cloning and Overexpression of 6-phosphate Sorbitol Dehydrogenase(gutF) from Lactobacillus Paracasei

为研究6-磷酸山梨醇脱氢酶基因在乳酸菌中超表达的影响,本试验根据副干酪乳杆菌的基因序列设计引物,用PCR的方法扩增6-磷酸山梨醇脱氢酶(gutF)基因,将其连接到乳酸菌表达载体pMG36e,并将重组质粒转化到副干酪乳杆菌及乳酸乳球菌中获得重组菌株;对重组菌株表达产物进行蛋白质水平的电泳检测,检测出表达了大约28kD的蛋白;使用山梨醇替代培养基中的葡萄糖,以未转化基因菌株做阴性对照,进行生长曲线的测定,结果表明,转化菌株能够利用在以山梨醇作为碳源的条件下,重组菌株的生长速度和最终菌浓度均优越于对照菌株。本研究通过超表达6-磷酸山梨醇脱氢酶蛋白,对于进一步研究乳酸菌的耐受性具有十分重要的理论基础。

This paper studied the overexpression of 6-phosphate sorbitol dehydrogenase gene in lactic acid bacteria. The 6-phosphate sorbitol dehydrogenase（gutF） gene was cloned by PCR method based on the gene of Lactobacillus paracasei. Specific gene was cloned into the lactic acid bacteria express vector pMG36e and the recombinant plasmid was electrotransformation into LactobaciUus paracasei L-14 and Lactococcus lactis LM0230 to get the recombinant strains. The results has showed about 28KD protein was expressed and detected in SDS- PAGE. The growth curve was determined with the medium in which sorbitol replaced glucose, and the strain without transforming gutF gene was choosed as control. The result showed recombinant strains can uselize sorbitol as carbon source, the growth rate and the final bacteria concentrations of recombinant strain were superior to control strains. The study of overexpression of 6-phosphate dehydrogenase might play a key role in exploring tolerance of lactic acid bacteria.