[1]崔苗苗,杨行洲,宋海艳,等.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,(05):867-874.[doi:doi:10.3969/j.issn.1000-4440.2025.05.005]
　CUI Miaomiao,YANG Xingzhou,SONG Haiyan,et al.Construction of a bacterial double-stranded RNA expression system targeting the trehalose-6-phosphate synthase genes of Aphelenchoides besseyi[J].,2025,(05):867-874.[doi:doi:10.3969/j.issn.1000-4440.2025.05.005]
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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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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2025年05期
页码:
867-874
栏目:
植物保护
出版日期:
2025-05-31

文章信息/Info

Title:
Construction of a bacterial double-stranded RNA expression system targeting the trehalose-6-phosphate synthase genes of Aphelenchoides besseyi
作者:
崔苗苗1; 2; 杨行洲1; 2; 宋海艳1; 2; 魏利辉2; 顾爱国3; 乐秀虎1; 李东霞1; 冯辉2
(1.河北工程大学园林与生态工程学院,河北邯郸056038；2.江苏省农业科学院植物保护研究所,江苏南京210014；3.江苏省产品质量监督检验研究院,江苏南京210007)
Author(s):
CUI Miaomiao1; 2; YANG Xingzhou 1; 2; SONG Haiyan1; 2; WEI Lihui2; GU Aiguo3; LE Xiuhu1; LI Dongxia1; FENG Hui2
(1.School of Landscape and Ecological Engineering, Hebei University of Engineering, Handan 056038, China;2.Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;3.Jiangsu Product Quality Testing & Inspection Institute, Nanjing 210007, China)
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
Keywords:
Aphelenchoides besseyi; trehalose-6-phosphate synthase genes; double stranded RNA; RNA interference
分类号:
S435.111.4+8
DOI:
doi:10.3969/j.issn.1000-4440.2025.05.005
文献标志码:
A
摘要:
外源双链RNA(dsRNA)可通过RNA干扰(RNAi)影响植物线虫的基因表达,达到杀虫防病的目的。利用细菌表达系统进行dsRNA制备,生产效率高、成本低,是dsRNA规模化制备的优选策略。为探索基于RNAi技术的细菌表达dsRNA在水稻干尖线虫(Aphelechoides besseyi)防治中的作用,本研究以水稻干尖线虫海藻糖6-磷酸合成酶基因AbTPS1和AbTPS2为靶标基因,设计构建细菌表达双链RNA(dsAbTPS1、dsAbTPS2)载体并转化大肠杆菌HT115(DE3),优化dsRNA诱导表达条件和提取方法,并利用荧光定量PCR测定细菌表达的dsRNA对靶标基因的沉默效率,评估其对水稻干尖线虫的防治效果。结果表明,当异丙基-β-D-硫代半乳糖苷(IPTG)浓度为0.5 mmol/L、诱导时间为6 h,本研究构建的携带AbTPS-L4440表达载体HT115菌株的dsRNA产量达到最大。利用Trizol法和甲酰胺法从表达菌株中提取的dsRNA产量显著高于乙醇-氯化钠法。用dsAbTPS1和dsAbTPS2处理水稻干尖线虫后,靶基因AbTPS1和AbTPS2表达水平下调,在4 ℃低温、40 ℃高温及脱水干燥环境中水稻干尖线虫存活率均显著低于M9缓冲液处理和dsGFP处理。本研究成功构建了水稻干尖线虫AbTPS1、AbTPS2基因的dsRNA细菌表达体系,获得的dsRNA具有明显的RNAi效应,为利用RNA农药防治水稻干尖线虫提供了技术支撑。
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 expression based on RNAi technology in the control of Aphelechoides besseyi, in this study, the trehalose-6-phosphate synthase genes AbTPS1 and AbTPS2 of Aphelechoides besseyi 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 methods 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 Aphelenchoides besseyi under low temperature of 4 ℃, high temperature of 40 ℃ and dehydration and drying conditions was significantly 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.

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备注/Memo

备注/Memo:
收稿日期：2024-09-30基金项目：江苏省农业科技自主创新基金项目[CX(22)3017]；河北省研究生创新资助项目(CXZZBS2024158)；邯郸市科学技术研究与发展计划项目(21422012329)作者简介：崔苗苗(1992-), 女, 河北邯郸人, 硕士研究生, 主要从事线虫防治研究。(E-mail)cuimiao0000@163.com通讯作者：乐秀虎,(E-mail)lexiuhu@163.com；冯辉,(E-mail)fenghui@jaas.ac.cn
更新日期/Last Update: 2025-06-24