【作者】 刘瑜；

【导师】 韩文君； 古静燕；

【作者基本信息】 山东大学 ， 生物与医药（专业学位）， 2023， 硕士

【摘要】 自放射性自显影技术被应用于埃希氏大肠杆菌(Escherichia coli)的基因组DNA观测以来,人们普遍认为细菌的基因组由一条单一环状染色体组成。然而,最近的基因组学研究表明,约10%的细菌含有多个复制子。有趣的是,已在许多资源菌株中发现了多复制子系统。随着基因组学的发展,细菌的多复制子系统必将被越来越多地识别和报告。然而,这种复杂遗传系统的形成机制及其存在意义尚不清楚,并且在一定程度上限制了人们对重要资源菌株的深度开发和精准应用。Persicobacter sp.JZB09是本课题组2009年自海岸沉积物中分离的一株多糖降解菌,目前已经在JZB09基因组中筛选、克隆了一系列高效的多糖降解酶资源。为进一步了解该菌株的基因组成、功能特色和生态意义,本研究经过二代结合三代测序,对JZB09菌株进行了基因组完成图的测定。本文发现:JZB09菌株拥有一个较大的细菌基因组,大小为8,257,130 bp;它是一个复杂的多复制子系统,由18个不同的复制环组成。因此,JZB09菌株是一个典型的多复制子系统的研究材料。本文进一步通过COG功能注释和CAZy功能注释等,对JZB09菌株不同复制子的功能差异进行了研究;还通过BLAST对JZB09不同复制子的近缘基因、共线性等进行了分析;本文还研究了 JZB09菌株的复制起始蛋白、分配系统蛋白等相关基因;并通过在线BLAST寻找了近缘菌株。本文通过比较基因组学,分析了 JZB09菌株与近缘种属菌株 Flammeovirga sp.MY04、F.kamogawensis str.YS10、F.yaeyamensis str.NBRC 100898及F.pectinis L12M1,在次级代谢途径上的异同,这五株菌都含有烯萜类次级代谢途径,推测可能合成类胡萝卜素导致菌株变橘黄(红)。最终,本文对JZB09和MY04菌株中典型的色素类次级代谢产物一类胡萝卜素进行了纯化、小量制备和质谱鉴定。本文的主要研究结果与分析如下:1、通过应用高通量测序技术(High-throughput sequencing)、拼装和注释,获得了JZB09的基因组完成图,并发现该菌株含有多复制子系统,适合深入分析。2、进一步分析了 JZB09菌株不同复制子的功能,结果显示:进行COG功能注释,大多数功能基因富集到Circle 1-Circle 6,且本文发现Circle 1的COG功能分类较为全面,这表明复制子Circle 1在维持JZB09基本生理功能方面很重要;而Circle 4-Circle 6以COG G(碳水化合物的运输和代谢)为主,这与JZB09是一株多糖降解菌相吻合;只有Circle 1-6、Circle 8和Circle 9注释到碳水化合物活性酶的功能模块,其中Circle 1以 Glycosyl Transferases(GT)为主,Circle2-6 以 Glycoside Hydrolases(GH)为主,这表明Circle 2-6对JZB09菌株降解中性多糖或碱性多糖具有重要作用;只有Circle 5和Circle 6上有Polysaccharide Lyases(PL)功能模块,这表明二者对于JZB09降解酸性多糖至关重要。因此,每个复制环扮演着不同的角色和功能。3、本文在JZB09的基因组中发现38个复制起始蛋白或分配系统的编码基因,并分布于复制子Circle 1-Circle 17中。这提示JZB09菌株的多复制子系统具有一定的稳定遗传能力,与课题组现有的遗传稳定性实验结果相吻合。4、次级代谢途径的比较分析显示:Persicobactersp.JZB09与近缘菌株Flammeovirga sp.MY04、YS10、NBRC100898和L12M1的基因组都编码烯萜(terpene)相关代谢途径,而类胡萝卜素就属于四烯萜类化合物,结合这5株菌的培养液在培养过程中有变红并伴有腥味的现象,本文推测这5株菌能够不同程度产生类胡萝卜素导致培养物变红。5、本文类胡萝卜素的纯化与鉴定实验显示:JZB09菌株和MY04菌株都能产生橘黄色色素,但Rf值不同。进一步经过HPLC纯化,二极管矩阵监测发现JZB09的产物有三个分离峰,而MY04菌株含有6个分离峰,其紫外-可见光扫描图显示这些峰均符合类胡萝卜素典型特征,并在400-500nm含有文献所述的经典肩峰。进一步的MS(质谱)鉴定实验表明,JZB09菌株中可产生包括ξ-胡萝卜素、链孢红素和β-胡萝卜素在内的类胡萝卜素。综上所述,本文完成了对Persicobacter sp.JZB09基因完成图的测定,并从不同方面深入比较了不同复制子的功能差异,还对JZB09的近缘菌株Flammeovirga sp.MY04、YS10、NBRC100898和L12M1进行了次级代谢的挖掘与比较研究,发现他们也都含有编码烯萜(terpene)生物合成途径功能蛋白的基因,负责合成类胡萝卜素等萜烯类多不饱和脂肪酸衍生物。因此推测:胡萝卜素类色素的产量与含量的变化,可能是引起桃色杆菌属、火色杆菌属菌株培养物颜色变化的原因。并且,本文还对类胡萝卜素进行了纯化、小量制备和结构鉴定,发现JZB09菌株和MY04菌株都能产生结构相似的胡萝卜素,但各自结构独特。本文不但对多复制子的研究提供了典型的细菌材料,也为资源菌株的深度开发和精确应用奠定了坚实基础。更多还原

【Abstract】 Since the application of autoradiography to the genomic DNA of Escherichia coli,it has been generally accepted that bacterial genomes usually consist of a single circular chromosome.However,recent genomic studies have shown that approximately 10%of bacteria contain multiple replicons.Interestingly,multiple replicon systems have been found in many resource strains,such as Brucella,Burkholderia,and Vibrio.With the development of genomics,bacterial multi-replicon systems will surely be increasingly identified and reportever,the formation mechanism and significance of this complex genetic system remain still unclear,which has limited the in-depth development and the precise application of important resource strains.Persicobacter sp.JZB09 was a polysaccharide-degrading bacterium isolated from the coastal sediments.A series of polysaccharide-degrading enzyme resources have been identified and cloned from the JZB09 genome.In order to further understand genetic composition,functional characteristics,and ecological significance of this strain,through the secondgeneration combined with the third-generation sequencing,the genome completion map of the strain JZB09 was determined.This study found that:the strain JZB09 has a large bacterial genome with the size of 8,257,130 bp,which is a complex multi-replicon system consisting of 18 different circular replicons.Therefore,the strain JZB09 is a typical research model of the multi-replicon system.This thesis further studied the functional differences of different replicons of strain JZB09 through COG functional annotation,CAZy functional annotation,etc.;through similar searches via BLAST,we analyzed the close genomes and the collinearity of different replicons of strain JZB09;we also studied the replication initiation protein,distribution system proteins and other related genes of strain JZB09 and searched for related strains through online BLAST.Through comparative genomics,the secondary metabolic pathway of strain JZB09 and closely related strains,e.g.Flammeovirga sp.MY04,F.kamogawensis strain YS10,F.yaeyamensis str.NBRC100898 and F.pectinis str.L12M1 were studied,and finally the typical pigment-the secondary metabolites-carotenoids were purified,prepared and structure-identified by the mass spectrometry.The main research results and corresponding analyses of this thesis are as follows:1.Through the application of high-throughput sequencing technology,assembly,and annotation,the complete genome map of JZB09 was obtained,and it was found that the strain contained a multi-replicon system,which was suitable for in-depth analysis.2.Further analyses of the functions of different replicons of the strain JZB09 showed that most of the COG functional annotations were enriched on Circle 1-Circle 6,and this thesis found that the COG functional classification of Circle 1 is relatively comprehensive,mainly including COG class J,which shows that Circle 1 maintains the basic functions of strain JZB09 aspect is very important;while Circle 4-Circle 6 is dominated by COG class G,which coincides with JZB09 being a polysaccharide-degrading bacterium.Only Circle 1-6,Circle 8,and Circle 9 were annotated to carbohydrate-active enzymes function modules,e.g.,Circle 1 was dominated by Glycosyl Transferases(GT),while Circle 2-6 were dominated by Glycoside Hydrolases(GH),which indicated that Circle 2-6 played an important role in the degradation of neutral or alkaline polysaccharides by strain JZB09,and only Polysaccharide Lyase(PL)function modules were found in Circle 5 and Circle 6,which suggested that they were very important for the strain JZB09 to degrade acidic polysaccharides.Therefore,these results suggested that each replication ring plays a different role and special function in bacterial degrading of polysaccharides.3.A total of thirty-eight genes that encoded replication initiation proteins and distribution systems were also found in the JZB09 genome,which were distributed in Circle 1-Circle 17.This suggests that the multi-replication subsystem of strain JZB09 has a certain stable genetic ability,which is consistent with the existed genetic experimental results by our research team.4.Analyses of secondary metabolic pathways have showed that the genome of Persicobacter sp.JZB09 and the near-source strains Flammeovirga sp.MY04,YS10,NBRC100898 and L12M1 all contained terpene-related pathways.Since carotenoids belonged to tetraterpenes we hypothesized that these five strains could all produce carotenoids and cause the culture color change(turn to red).5.The purification and identification experiments of carotenoids showed that both the strain JZB09 and the strain MY04 can produce orange-yellow pigment,and they both can produce carotenoids containing the same large class of carotenoids with similar polarity,but their Rf values are different;after the HPLC separation,it was found that the product of strain JZB09 has three different peaks,and the UV-visible full scan shows that these three peaks are in line with the typical characteristics of carotenoids:at 400-500nm,which contains the classic shoulder peaks described in the literature;The products by strain MY04 contains six kinds of peaks,and the UV-visible full scan shows that these six peaks are in line with the typical characteristics of carotenoids.The MS identification experiments indicated that the strain JZB09 can bio-synthesize ζ-carotene,neurosporene,and β-carotene.In summary,this thesis has obtained the complete the genome map of Persicobacter sp.JZB09,and compared the functional differences of each replicon in depth from many aspects.The secondary metabolisms of Flammeovirga sp.MY04,YS10,NBRC100898 and L12M1,which are closely related to the strain JZB09,were also analyzed and compared.It was found that they all contain terpene-related pathways,and their corresponding products might also contain carotenoids and other terpenes.Therefore,this thesis hypothesized that the changes in the production and content of carotene pigments may be the common cause of the color changes in the bacterial cultures of Persicobacter and Flammeovirga genera.Moreover,this thesis also carried out the purification,small-scale preparation,and structural identification of carotenoids,and we found that both strain JZB09 and strain MY04 can produce carotene with similar structures,but they each has own unique carotene.This research on the multiple replicons of stain JZB09 provided typical bacterial models and a solid foundation for the in-depth development and precise application of resource strains.更多还原