基于细菌表达dsRNA pool策略的贝类基因沉默方法OA

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.