渔业研究2025，Vol.47Issue(5)：640-650,11.DOI:10.14012/j.jfr.2025075
大牡蛎弧菌S2胶原酶基因的克隆、表达及其对菲律宾蛤仔基因表达的调控
Cloning and expression of the collagenase gene from Vibrio crassostreae S2 and its regulation on gene expression in Manila clam Ruditapes philippinarum
摘要
Abstract
[Objective]Manila clam(Ruditapes philippinarum)is a key aquaculture shellfish species in China,however,frequent bacterial diseases severely constrain its hatchery production.Vibrio crassostreae S2 is an im-portant pathogen during clam larval rearing.The purpose of this study is to identify the pathogenic factor of V.crassostreae and screen for chemicals that can inhibit the pathogenic factor.[Methods]The collagenase gene(colVc)was cloned using PCR with designed primers,ligated into the expression vector pET-28a(+),and ex-pressed in Escherichia coli BL21(DE3).Pathogenicity was assessed through injection assay,and transcriptomic data was used to reveal the regulation of collagenase on gene expression in clams.Small molecule inhibitors were screened,based on the ability to inhibit the enzymatic activity of collagenase.[Results]The colVc gene encoded a protein containing 277 amino acids,without the classic domain.The expression strain BL21/pET28colVc produced recombinant collagenase rColVc,which induced more than 20%mortality in clams upon injection.Transcriptomic data identified 132 differentially expressed genes(DEGs).The downregulated DEGs were enriched in BPs of DNA recombination,DNA integration,and DNA metabolic processes;MFs of actin binding and cytoskeletal protein binding.KEGG enrichment analysis showed that the downregulated DE-Gs in the collagenase-treated group of clam were primarily enriched in the actin cytoskeleton regulation path-way,implicating the critical role of collagenase in virulence.Finally,using the enzymatic activity of the colla-genase as the target,the compounds,riboflavin,curcumin,proanthocyanidins,palmatine hydrochloride,2'-hy-droxychalcone,chlorhexidine,and benzalkonium chloride were tested to exhibit inhibitory effects on enzymatic activity of collagenase.[Significance]The study is the first time to elucidate the pathogenicity of collagenase in V.crassostreae S2 and explore its regulation on gene expression of Manila clam,providing the potential tar-get for controlling vibriosis in clam hatcheries.关键词
菲律宾蛤仔/大牡蛎弧菌/胶原酶/基因表达Key words
Ruditapes philippinarum/Vibrio crassostreae/collagenase/gene expression分类
农业科技引用本文复制引用
穆志新,廖凯,张卫卫,徐继林..大牡蛎弧菌S2胶原酶基因的克隆、表达及其对菲律宾蛤仔基因表达的调控[J].渔业研究,2025,47(5):640-650,11.基金项目
国家重点研发计划项目(2024YFD2401700) （2024YFD2401700）
国家现代农业产业技术体系(CARS-49) （CARS-49）
