马奶酒样乳杆菌ZW3中WANG_1291基因的表达

【目的】马奶酒样乳杆菌ZW3含有一段长度为14.4 kb的胞外多糖合成基因簇，包含17个与胞外多糖合成相关的基因(WANG_1283?WANG_1299)，主要分析17个基因在马奶酒样乳杆菌ZW3生长过程中不同时间段的表达量，探究其中一个表达量发生变化的基因对乳酸菌产胞外多糖的影响。【方法】通过半定量RT-PCR实验，对基因簇上各基因的表达量进行分析；通过构建含有表达量变化基因的重组乳酸乳球菌，比较重组菌与野生菌的产胞外多糖差异。【结果】经分析，WANG_1284、WANG_1286、WANG_1287、WANG_1288、WANG_1290、WANG_1291、WANG_1292、WANG_1294、WANG_1296、WANG_1297、WANG_1298、WANG_1299这12个基因在菌体生长的50 h和60 h (产糖量上升阶段)表达量最高，推测这些基因在多糖聚合过程中起作用。从这12个基因中选出一个表达量发生明显变化的基因WANG_1291做进一步研究。将WANG_1291插入乳酸菌表达载体pMG36e中，构建了重组表达载体pMG36e-1291。将构建的重组表达载体转化到乳酸乳球菌WH-C1中，得到重组菌株。测定重组菌与野生菌生长特性，发现重组菌与野生菌之间的生长速度存在一定差异。然后利用苯酚-硫酸法测得重组乳酸乳球菌的胞外多糖产量是野生菌的2.1倍，胞外多糖产量有了明显的提高。【结论】确定WANG_1291基因是调控马奶酒样乳杆菌ZW3产胞外多糖的关键基因之一。

Expression of WANG_1291 gene of Lactobacillus kefiranofaciens ZW3

[Objective] The whole genome sequencing of Lactobacillus kefiranofaciens ZW3 is already known, it carries a 14.4 kb EPS (exopolysaccharides) gene cluster (WANG_1283 to WANG_1299) containing 17 EPS-related genes. In this study, we analyzed the expression quantity of 17 EPS genes in Lactobacillus kefiranofaciens ZW3 growth, and explored one expression quantity changed gene how to influence EPS yield. [Methods] Expression quantity of 17 EPS genes was analyzed by semi-quantitative RT-PCR technique; By constructing a recombinant strain of Lactococcus lactis containing an expression quantity changed gene, the EPS yield was compared between mutant strain and the wild strain. [Results] WANG_1284, WANG_1286, WANG_1287, WANG_1288, WANG_1290, WANG_1291, WANG_1292, WANG_1294, WANG_1296, WANG_1297, WANG_1298, WANG_1299 have the highest expression level at 50 h and 60 h when EPS was highly synthesized, suggesting that these genes play a role in the polymerization of polysaccharides. WANG_1291 was picked out from EPS genes to be further studied. This gene was inserted into plasmid pMG36e for the recombinant expression. The recombinant expression vectors were electroporated into Lactococcus lactis WH-C1. Assessing growth curves showed that the mutant strain and the wild strain have some differences. EPS yield of the recombinant was 2.1 times higher than that of the wild strain as analyzed by phenol-sulfuric acid method. [Conclusion] We provided evidence to show that WANG_1921 is a key gene to regulate Lactobacillus kefiranofaciens ZW3 EPS yield.