【作者】 孙志宏；

【导师】 张和平；

【作者基本信息】 内蒙古农业大学 ， 农产品加工及贮藏工程， 2014， 博士

【摘要】 Lactobacillus是乳酸菌最大的一个属,在人和动物胃肠道微生态平衡有着重要的作用,也是乳制品发酵剂以及益生菌产品的主要菌株,保加利亚乳杆菌更是乳制品发酵剂必不可少的菌种之一。系统解析乳杆菌属以及保加利亚乳杆菌的遗传背景和进化历程对开发利用乳杆菌有着极其重要的意义。本研究采用基因组重测序技术完成了146株Lactobacillus模式菌株基因组的测定,结合公共数据库中已完成的3株Lactobacillus模式菌株的全基因组序列,深入解析了149株Lactobacillus模式菌株的种系发育情况和物种分化历程,同时以305株L. delbrueckii subsp. bulgaricus分离株为研究对象,采用多位点序列分型技术对其进行微进化分析,得出如下结论：(1)完成了146株Lactobacillus模式菌株全基因组精细图的绘制,建立了含有72个基因的Lactobacillus核心基因集,大于70000个基因的泛基因集,结合基因组大小、GC含量、编码蛋白数量以及ANI和TNI值,显示出Lactobacillus基因组遗传多样性极其丰富,高于传统细菌分类学定义中的“科”。(2)基于基因组序列重构代表26个门、421个属的系统发育树,指出Oenococcus、 Pediococcus、Weissella、Leuconostoc和Lactobacillus属于一个进化分支Lactobacillus complex,而Lactococcus、Streptococcus和Enterococcus处于Lactobacillus complex分化前的另一个分支。(3)以72个核心基因为依据从基因组水平重构乳杆菌不同种的系统发育关系,所有乳杆菌模式菌株分为2个大的进化分支。进一步分析揭示了属于兼性异型发酵的乳杆菌模式菌株占据这一属的古老分支,即祖先位置,属于严格同型发酵、严格异型发酵的乳杆菌模式菌株则占据年轻分支,为后代菌株。同时发现处于第1进化分枝的动物源乳杆菌伴有生态位移动现象,第2进化分枝的动物源乳杆菌在动物生境和其他生境中呈现平行进化现象。(4)采用8个管家基因的多位点序列分型技术305株L. delbrueckii subsp. bulgaricus区分为121个ST型,组成14个同源复合体。通过STRUCTURE种群分析预测了可能存在5个主要的祖先群体,并推断蒙古国地区L. delbrueckii subsp. bulgaricus分离株更接近最早的祖先群体。(5)进一步分析表明L. delbrueckii subsp. bulgaricus祖先株与蒙古国地区分离株最接近,呈现从蒙古国地区向俄罗斯布里业特、图瓦、卡尔梅克和中国新疆、青海、西藏等地区传播的趋势。并指出L. delbrueckii subsp. bulgaricus不同序列型与其分离源、分离地有着直接的关系。更多还原

【Abstract】 Lactobacillus is the largest genus of lactic acid bacteria that plays important roles in balancing the microbiota of human and animal gastrointestinal tracts. They are the major dairy starter strains and probiotic products. Lactobacillus delbrueckii subsp. bulgaricus is one of the essential dairy starters used for manufacturing of fermented dairy products. Phylogenetic and species differentiation analysis is an extremely important for the development and utilization of Lactobacillus strains. In this study, the whole genomes of146Lactobacillus type strains were deciphered using whole-genome re-sequencing technology. Phylogenomic and evolutionary analysis of149Lactobacillus type strains was performed based on the whole genome level, which combined with the complete genome sequence of three Lactobacillus type strains. Simultaneously, the microevolution of305L. delbrueckii subsp. bulgaricus was determined using multi-locus sequence typing (MLST) technology. The results are as follow:(1) The whole genomes of146Lactobacillus type strains were deciphered. Only72homologous genes were found to be shared by all lactobacilli, and they were assigned to the core-genomes. More than70,000gene families were identified to pan-genomes, with its size growing continuously with the number of genomes. The results integrated with genome size, GC content, the number of protein-coding, ANI and TNI values, indicated an extremely high genetic diversity of the genomes of Lactobacillus. The genetic diversity is larger than typical families.(2) A phylogenetic tree with representative genomes of421genera from26phyla was reconstructed with Lactobacillus strains. The results showed that the Lactobacillus clade actually included strains from five genera, Oenococcus, Pediococcus, Weissella, Leuconostoc and Lactobacillus, thus, it was proposes that these five genera should be grouped under the Lactobacillus complex. In contrast, Lactococcus, Streptococcus, and Enterococcus, were located immediately before the Lactobacillus complex.(3) The phylogenetic tree of the Lactobacillus type strains was reconstructed based on the72core genes. All type strains of the Lactobacillus genus were divided into2big branches. Furthermore, the ancestral lineages in the phylogenetic tree were facultatively heterofermentative (FHE), which were at the beginning of the genus of Lactobacillus formation. In addition, the type strains, isolated from humans and animals, appeared to go through niche shift events on branch1but evolved parallelly with strains in other niches on branch2.(4) A total of305strains of L. delbrueckii subsp. bulgaricus were identified, belonging to121STs and14clonal complexes, using multilocus sequence typing (MLST) technology based on8housekeeping genes. Five ancestral lineages possibly existed among all isolates of L. delbrueckii subsp. bulgaricus by the analysis of STRUCTURE. Our results also revealed that the isolates from different regions in Mongolia were closer to ancestral lineages of L. delbrueckii subsp. bulgaricus.(5) The results further demonstrated that the isolates from different regions in Mongolia were the closest to the ancestral lineages of L. delbrueckii subsp. bulgaricus, which spreaded from Mongolia to Buryatia, Tuva, Kalmyki of Russia, and Xinjiang, Qinghai, Tibet of China. It was also demonstrated that there was a direct relationship between isolates of L. delbrueckii subsp. bulgaricus and their isolation location and origin.更多还原