诱变酒酒球菌中抗酸候选基因ATPase和FtsH的表达差异分析及功能验证

Differential Expression Analysis and Functional Verification of Acid-resistant Candidate Genes ATPase and FtsH in Mutagenized Oenococcus oeni

为研究胁迫条件下具有不同表达趋势的抗酸候选基因ATPase和FtsH是否可以提高菌株的抗酸性,利用实时荧光定量PCR(RT-qPCR)测定诱变酒酒球菌抗酸菌株b1和酸敏菌株b2中ATPase和FtsH在pH 4.8(最适条件)和pH 3.0(胁迫条件)ATB培养基中的相对表达量,分析其表达趋势;从b1中克隆ATPase和FtsH,分别命名为ATPase1和FtsH1,从b2中克隆ATPase和FtsH,分别命名为ATPase2和FtsH2,分析序列差异;将ATPase1,ATPase2,FtsH1和FtsH2分别在植物乳杆菌XJ25中做功能验证,构成重组植物乳杆菌a1,a2,f1和f2。以含空载体的植物乳杆菌con为对照,于pH 3.2的MRS培养基中测定重组植物乳杆菌的生长能力。定量结果表明ATPase在菌株b1和b2中表达量均显著上调;FtsH在b1中不变,在b2中显著上调。ATPase1和ATPase2在功能结构域内有3处非同义突变,FtsH1和FtsH2在跨膜结构域内有1处非同义突变。功能验证结果表明:a1和a2的生长能力均强于con,且a1和a2生长能力无差异;f1生长能力强于con,f2生长能力与con无差异。由此可知ATPase1,ATPase2和FtsH1均可以提高菌株抗酸性。

To investigate whether the acid-resistant candidate genes ATPase and FtsH with different expression trends under stress conditions can improve the acid resistance of strains,the real-time quantitative PCR(RT-qPCR)was used to determine the relative expression levels of mutagenized Oenococcus oeni,acid-resistant strain b1 and acid-sensitive strain b2,in ATB medium at pH 4.8(optimal condition)and pH 3.0(stress condition).Then their expression trends were analyzed.ATPase and FtsH were cloned from b1,which were named ATPase1 and FtsH1;ATPase and FtsH were cloned from b2,which were named ATPase2 and FtsH2.Next analyzed the sequence differences.The function of ATPase1,AT Pase2, FtsH1 and FtsH2 were verified in Lactobacillus plantarum (L. plantarum) XJ25, to establish recombinant L. plantarum a1, a2, f1 and f2. The growth ability of recombinant L. plantarum was determined in MRS medium at pH 3.2, using L. plantarum con containing empty vector as a control. Quantitative results showed that ATPase was significantly up-regulated in both strains b1 and b2;FtsH was unchanged in b1 and significantly up-regulated in b2. ATPase1 and ATPase2 have three non-synonymous mutations in the functional domain, and FtsH1 and FtsH2 have one non-synonymous mutation in the transmembrane domain. The functional verification results showed that the growth ability of a1 and a2 was stronger than con, and there was no difference in the growth ability between a1 and a2;the growth ability of f1 was stronger than that of con, and the growth ability of f2 was not different from that of con. It can be seen that ATPase1, ATPase2 and FtsH1 can all improve the acid resistance of the strain.