【作者】 王宇；

【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， 2020， 硕士

【摘要】 嗜热链球菌(Streptococcus thermophilus)作为自然发酵乳制品中的重要菌种,是工业生产发酵乳制品常用的发酵剂菌种之一,具有广泛的应用前景和经济价值。菌株的产酸、糖代谢和蛋白水解等生产性能与发酵乳的品质密切相关,因此,基于全基因组和甲基化信息解析嗜热链球菌基因型与表型之间的内在联系对于嗜热链球菌的开发利用意义重大。本研究以产酸性能差异较大的22株代表性嗜热链球菌菌株为研究对象,采用PacBio SMRT测序平台从全基因组和甲基化组水平探索菌株表型与基因型的内在关系。主要研究结果如下:(1)获得22株嗜热链球菌的全基因组完成图,发现不同菌株基因组序列高度保守,但与应激蛋白、生物合成和噬菌体相关的蛋白编码基因在菌株间有一定差异。(2)利用比较基因组学方法分析了 22株嗜热链球菌基因组中与产酸、蛋白水解、碳水化合物活性酶和CRISPR-Cas系统等相关的重要功能基因,发现相关功能基因的拷贝数在不同菌株间差异很小,且菌株中产酸功能基因的拷贝数与酸度值无显著相关性,但存在PrtS胞外蛋白酶的6株菌具有较强的产酸能力,都属于快产酸类群。(3)嗜热链球菌基因组中鉴定到m4C和m6A两种甲基化位点,其中m4C位点的平均甲基化水平高达60%,是嗜热链球菌中主要的甲基化修饰。每株嗜热链球菌中检测到2～8种甲基化motif基序,不同菌株间甲基化修饰模式呈现出高度多样性。(4)22株嗜热链球菌划分为快产酸类群和慢产酸类群两组,两类群之间共鉴定到3130个m4C和371个m6A差异甲基化基因,这些基因主要富集于氨酰tRNA生物合成、嘌呤代谢和萜类化合物骨架生物合成等代谢通路,表明这些代谢通路可能是调控嗜热链球菌产酸特性的主要因素,从表观遗传学角度揭示了 DNA甲基化对嗜热链球菌产酸特性的可能调控机制。综上所述,本研究从基因组学和甲基化组学水平揭示了嗜热链球菌基因型与表型之间的相关性。22株嗜热链球菌的全基因组序列高度保守,与表型特征相关的功能基因在不同菌株间差异很小,但快产酸和慢产酸类群间存在差异甲基化代谢通路,显示出DNA甲基化与嗜热链球菌菌株的表型特征有着密切联系。本研究有助于深入解析嗜热链球菌重要产酸特性形成的机理机制,为嗜热链球菌资源的利用和优良发酵剂的开发提供借鉴。更多还原

【Abstract】 As an important bacteria species in natural fermented milk,Streptococcus thermophilus(S.thermophilus)is one of the common leavening used in fermented dairy production and has extensive application prospect and economic value.The fermentation properties of the strain,such as acid production,glycometabolism and protein decomposition capacity are closely related to the quality of fermented milk.Therefore,it is of great significance for the development and utilization of S.thermophilus to analyze the internal relationship between the genotype and phenotype based on the whole genome and methylation information.In the present study,22 representative strains of S.thermophilus with significant differences in acid-producing performance were taken as the research objects.The PacBio SMRT sequencing platform was used to analyze the intrinsic relationship between genotype and phenotype of S.thermophilus at the genomes and methylomes level.The results were shown as follows:(1)The complete genome-wide map of 22 S.thermophilus strains were deciphered,these S.thermophilus genome sequences were highly conserved.However,the genes encoding stress proteins,biosynthesis,and phage related proteins are different among strains.(2)The important functional genes related to acid production,proteolytic ability,carbohydrate active enzyme and CRISPR-Cas system were analyzed by comparative genomics of 22 S.thermophilus.The copy numbers of these interrelated functional genes were little difference among strains,and there was no significant correlation between the copy numbers of acid producing functional genes and acidity values.However,the six strains with the extracellular protease(PrtS)had strong acid-producing ability,all of which belonged to the fast acid-producing group.(3)Two methylation patterns of m6A and m4C were identified in 22 S.thermophilus.The average methylation level of m4C sites was as high as 60%,which is the main methylation modification in S.thermophilus.In addition,2 to 8 motifs of the methylated sites were detected in each strain of S.thermophilus,indicating a high diversity of methylation modification patterns among different strains.(4)Twenty-two S.thermophilus genomes were divided into two groups:fast acid-producing and slow acid-producing,a total of 3130 m4C and 371 m6A differentially methylated genes were identified between the two groups,respectively.These genes are significantly enriched in metabolic pathways such as aminoacyl-tRNA biosynthesis,purine metabolism and terpenoid backbone biosynthesis,of which play an important role in the acid production of S.thermophilus.The possible regulatory mechanism of DNA methylation effect on the acid-producing ability of S.thermophilus is revealed from the perspective of epigenetics.In summary,this study analyzed the correlation between the genotype and phenotype of S.thermophilus based on genomics and methylomes.The whole genomes of 22 S.thermophilus are highly conversed,and the differences were little of key functional genes affecting phenotype characteristics among different strains.Whereas there were differential methylation metabolic pathways between fast acid-producing and slow acid-producing strains,showing DNA methylation was closely related to the phenotypes of S.thermophilus.This study was conductive to analyze the formation mechanism of important acid-producing properties of S.thermophilus,and provided a reference for the resources utilization of S.thermophilus and the development of excellent starter culture.更多还原