水稻干尖线虫海藻糖6-磷酸合成酶基因双链RNA细菌表达体系构建

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关键词：水稻干尖线虫；海藻糖-6磷酸合成酶基因；双链RNA；RNA干扰中图分类号： S435.114+8 （204号 文献标识码：A 文章编号：1000-4440（2025）05-0867-08

Abstract： Exogenous double-stranded RNA （dsRNA）can affct the gene expression of plant nematodes through RNAinterference（RNAi）to achieve the purpose ofpest control. The use of bacterial expression system for dsRNA preparation has high production efficiency and low cost， whichis the preferred strategy forlarge-scale preparation of dsRNA.Inorder toexplore theroleofbacterial dsRNA expression based on RNAi technology in the control of Aph 1 elechoidesbesseyi，in thisstudy，the trehalose-6-phosphate synthase genes AbTPS1 and AbTPS2of Aphelechoidesbes

seyi wereusedastarget genes to designand constructbacterial expressiondouble-strandedRNA（dsAbTPS1 and dsAbTPS2）vectorsandtransform Escherichiacoli HTi15（DE3）.Theinduction expresionconditionsand extractionmethodsof dsRNA wereoptimized，andthe silencing eficiencyof dsRNA expressedbybacteriaontarget genes wasdetermined byfluorescence quantitative PCR，and the control effct of dsRNAonAphelenchoides beseyi was evaluated.The results showed that when the concentration of isopropyl ⋅β D -thiogalactoside（IPTG）was O.5 mmol/L and the induction time was 6 h ，thedsRNA yieldofthe AbTPS -L4440 expression vector HT115 strain constructed in this study reached the maximum. TheyieldofdsRNA extractedfromtheexpresionstrainsusing Trizolmethodandformamide method was significantlyhigher thanthatobtainedusing ethanol-sodiumchloride method.After treating Aphelenchoidesbeseyi with dsAbTPS1 and dsAbTPS2，the expresion levels of target genes AbTPS1and AbTPS2 were down-regulated.The survival rate of Aphelenchoidesbesseyi underlow temperature of 4 C ，high temperature of 40 ΔC and dehydration and drying conditions was significantlylowerthan thatinM9buffertreatmentanddsGFP treatment.Inthisstudy，thebacterialdsRNAexpresionsystem targeting AbTPS1 and AbTPS2 of A . besseyi was successfully constructed，and the obtained dsRNA exhibited obvious RNAi effects，which provided technical support for the use of RNA-based pesticides to control A .besseyi.

Key Words：Aphelenchoides besseyi； trehalose-6-phosphate synthase genes；double stranded RNA；RNA interferen

RNA干扰（RNAinterference，RNAi）是一种进化上保守的序列特异机制，能调控大部分真核生物基因表达。（剩余12695字）