【作者】 李贵阳；

【导师】 莫照兰；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2011， 博士

【摘要】 鳗弧菌是水产病害中最常见的细菌病原,可感染鱼虾贝等多种水生动物,对养殖业造成巨大经济损失。鳗弧菌的基因组尚未阐明。本文开展了临床致病鳗弧菌株M3(血清型O1)和非致病标准菌株ATCC43308(血清型O4)的全基因组序列测定、基因组和转录组比较分析,为从整体了解鳗弧菌的生态、生理及致病机制打下基础。首先用Roche 454 FLX Titanium测序得到的序列信息进行基因组骨架组装,然后用AB SOLID测序得到的序列信息进行高覆盖度测序补洞、scaffold搭建及延伸,完成拼接组装,对所获DNA序列进行了全面的生物信息学分析。分析结果显示:M3形成了23个scaffold,contig N50长度为334.6kb,基因组总G+C%含量为44.5%,预测并注释成功的CDS(coding sequence)总数为4009个,CDS平均长度为295aa;含48个tRNA和3个16s-23s-5s rRNA。ATCC43308有15个scaffold,N50长度为240.6 kb,基因组总G+C%含量为44.4%,有3820个注释过的CDS,平均长度为293aa,含有49个tRNA和3个16s-23s-5s rRNA。两株菌中注释的约27%CDS都是参与细胞的生命运动代谢过程,包括氨基酸转运和代谢,糖脂的代谢,DNA复制与修复,RNA转录与调控等;约10%CDS与毒力相关,包括粘附素,菌毛,分泌系统和转运系统等。两株菌存在较丰富的水平转移遗传元件,在M3和ATCC43308分别发现28和14个插入序列(IS)、21和19个Tn及18和9个prophage序列成份。在详细分析两株菌基因组特征基础上,对它们进行了比较基因组研究,发现了鳗弧菌的代谢机制及致病性/毒力强弱的特征。M3基因组比ATCC43308大约230kb左右,而M3注释的CDS比ATCC43308多190个。其中差异基因较多的主要集中在参与信号传导过程、酶代谢、细胞菌膜形成和外膜蛋白以及假定蛋白和COG未包含的蛋白方面。两株菌中均发现了新的溶血素和金属蛋白酶及与T6SS相关基因。结果表明,ATCC43308缺失了荚膜多糖转运系统基因簇wza-wzb-wzc,使得荚膜多糖不能正常输送到胞外;M3中存在更多的有激活转录成份的IS序列,有利于更多基因的表达,并且在M3中发现了T3SS成份,而这些都与细菌的毒性密切相关,推测可能与M3致病性有关。在转录组测序方面,对M3和ATCC43308基因表达量进行了比较,发现了191个表达有显著差异的基因,其中也包含与致病性相关的毒力因子。由于鳗弧菌对养殖的严重危害,已有很多方法对其进行检测,如荧光PCR,免疫探针,血清学等。本文根据M3和ATCC43308以及NCBI上已公布的弧菌属全基因组序列,利用toxR-toxS两成份系统序列保守性设计了引物,建立了针对鳗弧菌、溶藻胶弧菌、哈氏弧菌、副溶血弧菌、创伤弧菌和霍乱弧菌六种水产常见病原微生物的多重PCR快速检测方法。结果表明,多重PCR引物特异性强,与其他弧菌及非弧菌无交叉反应,检测灵敏度为10-100 CFU,同时也对临床病料进行了检测,证明该多重PCR效果确实。更多还原

【Abstract】 Vibrio anguillarum is a Gram-negative Gammaproteobacteria and can cause epidemic vibriosis in fish, bivalves and crustaceans, with the character as a hemorrhage septicemia to beget enormous economic losses. Whole genome sequencing of Vibrio anguillarum clinical pathogenic strain M3 and nonpathogenic ATCC43308 were perfomed with a combined strategy of using Roche 454 GS(FLX Titanium) pyrosequencing and AB SOLID mate-paired sequencing technology. In addition, based on the genomic information, transcriptomic profilings of M3 and ATCC43308 were analyzed by RNA-Seq tech. The sequence analysis of genome and transcriptome provided molecular-level understanding of genetic information and pathogenicity about Vibrio anguillarum.Pathogenic M3 was made of 23 scaffolds, with the length of N50 with 240.6 kb, the content of G+C% with 44.5%, the amount of predicted and annotated CDS with 4009 and the average length of CDS with 295aa, with 48 tRNAs and 3 16s-23s-5s rRNAs. For nonpathgenic ATCC43308 with 15 scaffolds, the length of N50 334.6 kb, the G+C% 44.4%, CDS with 3820, the average length of CDS with 293aa, 49 tRNAs and 3 16s-23s-5s rRNAs. 27% of CDS participated in the metabolism, transport and replicate, transcription regulation et al. Some(about 10% CDS)were responsible for the pathogenesis containing secretion, adhesin and pilus et al.Comparative genomics analysis among M3, ATCC43308 and other related available species described the detailed characteristics of the metabolism mechanism and pathogenesis about V. anguillarum. The strain ATCC43308 lacked a gene cluster, wza-wzb-wzc, which is responsible for the capsular polysaccharides transport through the cell membrane. Compared to ATCC43308, M3 were larger about 230 kb in genome seize, more 190 CDS in amount. The differences mainly pooled in the aspects including signal transduct, enzyme metabolism, cell envelope biogenesis and function unkown. For M3 and ATCC43308, more new hemolysin、metalloprotease and T6SS-related genes were found, some of which respected to the virulence of pathogen. Furthermore, compared to ATCC43308, more exterior sequences such as IS (28 vs 14) ,Tn(21 vs 19)and prophag(e18 vs 9), some of which are proved to be activators of other genes, were found in M3. We speculated that the components were necessary for M3 to cause diseases.Comparative transcriptomic analysis of the two strains revealed 191 genes whose expression level differentiated remarkably.Many methods were perfomed to detect the pathogen, such as real time PCR, immunoprobe and serological reaction. Based on the genome sequences of the two-component system toxR-toxS of genus Vibrio, six pairs of conserved primers to simultaneously detect main pathogenic vibrio including V. anguillarum, V. alginolyticus, V. harveyi, V. parahaemolyticus, V. vulnificus and V. cholerae were designed. The results showed that multiplex PCR have fairly specific to Vibrio, and nonspecific and cross-reaction were not found. Reaction sensitivity was 10~100 CFU. The detection for real samples also proved its reliability.更多还原