水生生物学报2026，Vol.50Issue(7)：149-156,8.DOI:10.3724/1000-3207.2025.2025.0447
基于细菌表达dsRNA pool策略的贝类基因沉默方法
DEVELOPMENT OF A GENE SILENCING METHOD FOR BIVALVE BASED ON BACTERIA-EXPRESSED DSRNA POOL STRATEGY
摘要
Abstract
RNA interference(RNAi)represents a promising technique for gene functional studies,yet its implementa-tion in bivalve mollusks has been hindered by two persistent obstacles:inefficient in vivo delivery and substantial off-target effects.To overcome these challenges,we developed an integrated RNAi strategy combining bacterially expressed double-stranded RNA(dsRNA)with a pooled dsRNA approach and applied it to silence the nuclear receptor nr5a2 gene in the zhikong scallop(Chlamys farreri).Five non-overlapping dsRNA fragments targeting the nr5a2 open reading frame were expressed in RNase III-deficient Escherichia coli HT115 and delivered orally via an established Spirulina-based feeding system.Comparative assessment revealed that feeding achieved higher and more prolonged nr5a2 knockdown than intramuscular injection,with 60.5%silencing on day 1 and remaining 33.4%on day 7.Tempo-ral profiling indicated effective silencing for up to 12h post-feeding,supported by bacterial viability assays that justi-fied a 12h feeding interval.Crucially,the dsRNA pool strategy minimized off-target effects:in silico analysis identi-fied only 110 potential off-target transcripts,and RNA-seq validation showed merely 4(females)and 8(males)with modest expression changes,none of which exhibited typical RNAi suppression patterns.In summary,we present a scala-ble,cost-effective RNAi platform that combines bacterial dsRNA production,algal delivery,and a pooled dsRNA design.This system achieves efficient(>50%)and specific gene silencing in a bivalve species,offering a robust tool for functional genomics and potential applications in aquaculture breeding.关键词
nr5a2/RNA干扰/细菌dsRNA表达/dsRNA pool/栉孔扇贝Key words
nr5a2/RNAi/Bacterial dsRNA expression/dsRNA pool/Chlamys farreri分类
农业科技引用本文复制引用
刘亚,韩莲雪,温玉欣,付慧,苗晶晶,潘鲁青..基于细菌表达dsRNA pool策略的贝类基因沉默方法[J].水生生物学报,2026,50(7):149-156,8.基金项目
国家自然科学基金(32473140)资助[Supported by the National Natural Science Foundation of China(32473140)] （32473140）
