河蟹螺原体非编码RNA及其毒力靶标的多组学系统挖掘

Multi-omics Analysis of Spiroplasma eriocheiris Non-coding RNAs(ncRNAs) and Their Virulence Targets

我国是世界最大水产养殖国,每年甲壳动物因病害造成的经济损失约为70亿元。其中,螺原体(Spiroplasma)是甲壳动物重要的致病菌之一,可造成虾蟹大面积死亡,已列入农业农村部三类疫病。非编码RNA(ncRNA)广泛存在于细菌中,其主要通过碱基配对识别靶标mRNA在转录后水平调节基因的表达,部分ncRNAs通过与蛋白质相互作用而影响蛋白质功能。近年研究表明,细菌ncRNAs在毒力调控中扮演极为重要的角色。为了研究河蟹螺原体ncRNAs在甲壳动物致病中的分子调控作用,需系统筛选鉴定螺原体感染相关的ncRNAs和毒力靶标。通过比较基因组、差异转录组、定量蛋白质组、系统生物学和分子相互作用联合研究得到:整合基因组和转录组挖掘得到河蟹螺原体ncRNAs共54个;在体内感染和体外培养的不同时期,利用数字基因表达谱分析分别得到11个和28个差异显著ncRNAs;利用4款生物软件预测ncRNAs靶标,取交集得到423个;利用定量蛋白质组检测,鉴定出68个差异毒力蛋白,这些差异毒力蛋白与ncRNAs的30个毒力靶标中的21个相同;利用网络生物学分析得到主要的节点Hub-ncRNA共有6个;利用RNA pull-down、原核链特异性测序和LC-MS/MS综合分析,得到重要节点ncRNA SR05的互作RNA 53个、互作蛋白质120个。相关研究成果,可为诠释河蟹螺原体致病机制及其与宿主相互作用机制奠定基础,为虾蟹该疾病的综合防治提供科学依据。

China is the one of the largest aquaculture countries in the world,and the annual economic loss caused by diseases of crustaceans is about 7 billion RMB per year.Among them,Spiroplasma is one of the important pathogenic bacteria of crustaceans,which can cause large-scale death of crustaceans,and has been listed as a Class Ⅲ epidemic disease of the Ministry of Agriculture.Non coding RNAs(ncRNAs)are widespread in bacteria.They recognize target mRNAs mainly through base pairing and regulate gene expression at the post transcriptional level.Some ncRNAs affect protein function by interacting with proteins.Recent studies have shown that bacterial ncRNAs play an important role in virulence regulation.In order to study the molecular regulatory role of Spiroplasma ncRNAs in crustaceans,it is necessary to systematically screen and identify the ncRNAs and virulence targets related to Spiroplasma infection.Through comparative genome,differential transcriptome,quantitative proteome,system biology analysis,and combined analysis of molecular interaction and bioinformatics methods,this study showed that 54 ncRNAs of Spiroplasma were obtained by integrating genome and transcriptome.Under the conditions of infection in vivo and culture in vitro,11 and 28 significantly different ncRNAs were obtained by digital gene expression profiling.Four biological software tools were used to predict ncRNA targets,and 423 targets were obtained by intersection.Using quantitative proteomic analysis,68 differential virulence proteins were identified,which were the same as 21 out of the 30 virulence targets of ncRNAs.Six main hub-ncRNAs were found by network biology analysis.Using RNA pull-down,prokaryotic chain specific sequencing and LC-MS/MS comprehensive analysis,53 interacting RNAs and 120 interacting proteins of ncRNA SR05 were found.The relevant research results can lay a foundation for interpreting the pathogenic mechanism of Spiroplasma and its interaction mechanism with the host,and provide a scientific basis for the comprehensive prevention and treatment of this disease in crustaceans.