耐酸与酸敏酒酒球菌环丙烷脂肪酸合酶基因的差异  

The Difference of Cyclopropane Fatty Acid Gene in Acid-resistant and Acid-sensitive Oenococcus oeni

作　　者：田雨[1] 陈其玲[1] 刘树文[1,2,3] 何玲 Tian Yu;Chen Qiling;Liu Shuwen;He Ling(College of Enology,Northwest A&F University,Yangling 712100,Shaanxi;Shaanxi Engineering Research Center for Viti-Viniculture,Yangling 712100,Shaanxi;Heyang Experimental Demonstration Station,Northwest A&F University,Weinan 715300,Shaanxi;College of Horticulture,Northwest A&F University,Yangling 712100,Shaanxi)

机构地区：[1]西北农林科技大学葡萄酒学院,陕西杨凌712100 [2]陕西省葡萄与葡萄酒工程技术研究中心,陕西杨凌712100 [3]西北农林科技大学合阳葡萄试验示范站,陕西渭南715300 [4]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《中国食品学报》2019年第7期64-71,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：宁夏回族自治区科技重大专项(2016BZ0603);“十三五”国家重点研发计划项目(2016YFD0400504-01);国家现代农业(葡萄)产业技术体系建设专项(CARS-29-jg-03)

摘　　要：目的:研究筛选得到的野生耐酸与酸敏酒酒球菌环丙烷脂肪酸基因(cfa)的差异,探索酒酒球菌环丙烷脂肪酸基因与酒酒球菌耐酸性的相关性。方法:35株野生酒酒球菌中,利用酸胁迫环境筛选耐酸能力最强的菌株,并与野生酸敏菌SX-1b及实验室离子注入诱变的耐酸、酸敏突变菌a3、b2共同进行环丙烷脂肪酸基因cfa的克隆、测序和比对;选择存在氨基酸突变的野生耐酸酒酒球菌及野生酸敏菌株的cfa基因在植物乳杆菌ATCC33222中进行外源表达,从而验证耐酸菌株cfa基因与耐酸能力的相关性。结果:(1)筛选出野生型耐酸酒酒球菌CS-7b和ME-5b,确定其耐酸生长极限为pH2.6;(2)对野生菌与诱变菌的cfa基因进行测序比对发现,耐酸菌株的野生型和诱变型的序列相同,较酒酒球菌PSU-1发生3处碱基突变,而酸敏菌野生型、诱变型的序列则与酒酒球菌PSU-1一致;(3)将SX-1b与CS-7b的cfa基因在植物乳杆菌中表达,构建重组载体pMG36e-cfa1和pMG36e-cfa2,并得到对应重组菌L1和L2;(4)在pH3.6培养基中培养,重组菌L1和L2的生长情况明显优于含空载体的植物乳杆菌L0,且L2的生长优于L1。而在pH3.4培养基中培养,L0没有生长迹象,只有重组菌L1和L2正常生长,cfa基因的导入突破了植物乳杆菌宿主菌的生长极限。结论:酒酒球菌cfa基因及基因间存在的稳定差异与菌株耐酸表型具有一定相关性。Objective: To explore the relationship between strains’ acid resistance and the cyclopropane fatty acid gene, this research was designed to study the difference in cfa gene between selected wild acid-resistant Oenococcus oeni strains and acid-sensitive strains. Methods: Thirty-five wild Oenococcus oeni were selected in the medium with low pH for the most acid-resistant strain. With acid-sensitive strain-SX-1b, the cfa genes of wild acid-resistant strains were cloned, sequenced and compared with acid-resistant mutant a3 and acid-sensitive mutant b2. The cfa genes of acid-sensitive strain and wild acid-resistant strain with amino acid mutations were expressed in Lactobacillus plantarum ATCC33222 to verify the correlation between cfa gene mutation of acid-resistant strains and strains’ acid resistance. Results: Oenococcus oeni CS-7b and ME-5b were selected as wild acid-resistant strains and the acid limit of their growth is pH 2.6;In the comparison of the sequence of cfa genes in wild strains and mutants, the sequence of wild and mutagenesis acid-resistant strains was identical and had mutations in three sites compared with Oenococcus oeni PSU-1. The sequences of wild and mutagenesis acid-sensitive strains were consistent with Oenococcus oeni PSU-1. The cfa genes of SX-1b and CS-7b were expressed in Lactobacillus plantarum ATCC33222. The recombinant plasmids pMG36e-cfa1 and pMG36e-cfa2 were constructed and corresponding recombinant strains-L1 and L2 were obtained. In the medium at pH 3.6, the growth of recombinant strais-L1 and L2 was significantly better than that of Lactobacillus plantarum L0 with empty vector, and the growth of L2 was better than L1. In the pH 3.4 medium, only the L1 and L2 grew up normally while L0 did not grow, which suggests the introduction of cfa gene broke the growth limit of Lactobacillus plantarum in acid resistance. Conclusion: The cfa gene in O. oeni strains and its stable mutation are related to the acid resistance ability.

关 键 词：酒酒球菌 耐酸性 环丙烷脂肪酸基因 外源表达 

分 类 号：TS262.6[轻工技术与工程—发酵工程]