江苏农业学报2025，Vol.41Issue(5)：867-874,8.DOI:10.3969/j.issn.1000-4440.2025.05.005
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
Construction of a bacterial double-stranded RNA expression system targeting the trehalose-6-phosphate synthase genes of Aphelenchoides besseyi
摘要
Abstract
Exogenous double-stranded RNA(dsRNA)can affect the gene expression of plant nematodes through RNA interference(RNAi)to achieve the purpose of pest control.The use of bacterial expression system for dsRNA preparation has high production efficiency and low cost,which is the preferred strategy for large-scale preparation of dsRNA.In order to explore the role of bacterial dsRNA ex-pression based on RNAi technology in the control of Aph-elechoides besseyi,in this study,the trehalose-6-phosphate synthase genes AbTPS1 and AbTPS2 of Aphelechoides bes-seyi were used as target genes to design and construct bacterial expression double-stranded RNA(dsAbTPS1 and dsAbTPS2)vectors and transform Escherichia coli HT115(DE3).The induction expression conditions and extraction meth-ods of dsRNA were optimized,and the silencing efficiency of dsRNA expressed by bacteria on target genes was determined by fluorescence quantitative PCR,and the control effect of dsRNA on Aphelenchoides besseyi was evaluated.The results showed that when the concentration of isopropyl-β-D-thiogalactoside(IPTG)was 0.5 mmol/L and the induction time was 6 h,the dsRNA yield of the AbTPS-L4440 expression vector HT115 strain constructed in this study reached the maximum.The yield of dsRNA extracted from the expression strains using Trizol method and formamide method was significantly higher than that obtained using ethanol-sodium chloride method.After treating Aphelenchoides besseyi with dsAbTPS1 and dsAbTPS2,the expression levels of target genes AbTPS1 and AbTPS2 were down-regulated.The survival rate of Aphelench-oides besseyi under low temperature of 4℃,high temperature of 40℃and dehydration and drying conditions was signifi-cantly lower than that in M9 buffer treatment and dsGFP treatment.In this study,the bacterial dsRNA expression system 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.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Key words
Aphelenchoides besseyi/trehalose-6-phosphate synthase genes/double stranded RNA/RNA interference分类
农业科技引用本文复制引用
崔苗苗,杨行洲,宋海艳,魏利辉,顾爱国,乐秀虎,李东霞,冯辉..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[J].江苏农业学报,2025,41(5):867-874,8.基金项目
江苏省农业科技自主创新基金项目[CX(22)3017] （22）
河北省研究生创新资助项目(CXZZBS2024158) （CXZZBS2024158）
邯郸市科学技术研究与发展计划项目(21422012329) （21422012329）
