Page 109 - 《渔业研究》2025年第5期

650 渔 业 研 究 第 47 卷

Cloning and expression of the collagenase gene from Vibrio crassostreae S2 and
its regulation on gene expression in Manila clam Ruditapes philippinarum

*
MU Zhixin，LIAO Kai，ZHANG Weiwei ，XU Jilin *
(School of Marine Sciences, Ningbo University, Ningbo 315832, China)

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
(col ) was cloned using PCR with designed primers, ligated into the expression vector pET-28a(+), and ex-
Vc
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 col gene
Vc
encoded a protein containing 277 amino acids, without the classic domain. The expression strain
BL21/pET28col produced recombinant collagenase rCol , which induced more than 20% mortality in clams
Vc
Vc
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.
Key words: Ruditapes philippinarum; Vibrio crassostreae; collagenase; gene expression