【摘要】 粪肠球菌是乳酸菌的一个属.采用生物信息学方法,预测了粪肠球菌AS1.2984基因组中乳酸脱氢酶和三磷酸甘油醛脱氢酶的组成型启动子序列.设计引物通过PCR扩增得到这2段启动子序列,并克隆到乳酸菌 大肠杆菌穿梭质粒pNZ8037上,在启动子下游接上绿色荧光蛋白基因作为标记,构建乳酸菌的组成型蛋白表达质粒.通过电转化将上述改造过的pNZ8037质粒导入粪肠球菌中,在共聚焦显微镜中可以观察到绿色荧光蛋白的表达.在乳酸菌MRS培养基中加入乳酸、盐酸、氢氧化钠等试剂,发现可以调节绿色荧光蛋白的表达量.更多还原

【Abstract】 Enterococcus facalis is one kind of lactic acid bacteria (LAB). By using bioinformatics, constitutive promoters of L-lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase were predicted from the genome of Enterococcus facalis AS1.2984. After designing 5′and 3′primers, the sequences of these two promoters were obtained by PCR. Then expression plasmids pGAP-GFP-pNZ8037 and pLDH-GFP-pNZ8037 were constructed with reporting protein-GFP just under the control of these two promoters in the shuttle plasmid-pNZ8037, and they were transformed into Enterococcus facalis by electric pulse. The expression of GFP is confirmed by confocus microscope. The expression of GFP is different at the existence of some reagents such as lactic acid, HCl and NaOH. So it is assumed that acidic and alkaline material can regulate these two promoters. These results show that more powerful promoters can be found in the LAB’s genome by bioinformatics in the short time.更多还原