甜菜夜蛾卵黄原蛋白受体基因的RNA干扰  

RNA Interference of Vitellogenin Receptor Gene in Beet Armyworm(Spodoptera exigua)

作　　者：赵静[1,2] 陶蓉[1] 郝德君 肖留斌[2] 谭永安[2] ZHAO Jing;TAO Rong;HAO DeJun;XIAO LiuBin;TAN YongAn(Co-Innovation Center for the Sustainable Forestry in Southern China/College of Forestry,Nanjing Forestry University,Nanjing 210037;Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014)

机构地区：[1]南京林业大学南方现代林业协同创新中心/林学院,南京210037 [2]江苏省农业科学院植物保护研究所,南京210014

出　　处：《中国农业科学》2019年第1期56-64,共9页Scientia Agricultura Sinica

基　　金：国家重点研发计划(6111661);国家现代农业产业技术体系(CARS-18-16);江苏省农业科学院院基金计划(6111612;6111613)

摘　　要：【目的】卵黄原蛋白受体(vitellogenin receptor,VgR)是介导昆虫卵黄原蛋白胞吞作用的主要受体,通过RNA干扰(RNAi)方法研究甜菜夜蛾(Spodoptera exigua)VgR的功能,为深入了解甜菜夜蛾生殖生理的分子机制及开发有效防治新方法提供依据。【方法】以甜菜夜蛾雌成虫腹部的cDNA为模板,通过PCR克隆得到甜菜夜蛾VgR基因片段,其包含配体结合域功能区。绿色荧光蛋白基因(GFP)片段通过特异性引物从笔者实验室保存的GFP质粒上扩增。将VgR和GFP目的片段连入pMD-19T载体后进行测序,利用DNAMAN软件分析基因序列的准确性。以测序验证正确的质粒作为DNA模板,利用带T7启动子的引物进行PCR扩增。用T7 RiboMAXTM Express RNAi System合成试剂盒合成VgR-dsRNA和GFP-dsRNA。应用10μL微量进样器在化蛹第2、6天的甜菜夜蛾雌蛹腹部注射3μL双链RNA(2μg·μL-1)。利用RT-qPCR技术检测甜菜夜蛾刚羽化、羽化24 h、羽化48 h雌成虫的VgR表达量变化,同时统计对照组(空白对照、注射GFP-dsRNA)和处理组(注射VgR-dsRNA)甜菜夜蛾的羽化率及单雌产卵量。【结果】扩增得到VgR和GFP基因片段,大小分别为327和417 bp。RT-qPCR检测结果表明,与对照组相比,注射dsRNA后甜菜夜蛾的VgR表达水平显著下降。对于刚羽化、羽化24 h、羽化48 h的雌成虫,注射VgR-dsRNA处理组的VgR表达量相比注射GFP-dsRNA的对照组分别下降了79.35%、84.22%、67.68%。通过解剖观察刚羽化、羽化24 h、羽化48 h的甜菜夜蛾卵巢,发现注射VgR-dsRNA处理组与注射GFP-dsRNA对照组相比,卵巢发育进度显著推迟。对于羽化24 h的甜菜夜蛾,与注射GFP-dsRNA组相比,注射VgR-dsRNA处理组卵巢管长度下降了23.92%;注射GFP-dsRNA组的卵巢成熟卵粒较多,平均直径为(0.46±0.05)mm,而注射VgR-dsRNA处理组成熟卵粒数量较少且相对较小,平均直径为(0.23±0.02)mm。注射GFP-dsRNA组和VgR-dsRNA组的羽化率无显著差异。注射VgR-ds【Objective】Vitellogenin receptor(VgR) is the main receptor that mediates the endocytosis of insect vitellin. The objective of this study is to clarify the function of VgR of beet armyworm(Spodoptera exigua) through RNA interference(RNAi) method, and to provide a basis for further understanding the molecular mechanism of reproductive physiology and developing effective new methods for prevention and control.【Method】The fragment of Vg R was amplified from the cDNA of female adults abdomen tissues of S. exigua by PCR which included the ligand-binding domain region. The green fluorescent protein gene(GFP) fragment was amplified from the GFP plasmid stored in the laboratory by specific primers. The fragment of VgR and GFP was then inserted into the pMD-19 T for sequencing. The nucleic acid sequence was analyzed by DNAMAN software. The correct plasmid confirmed by sequencing acted as the DNA template. PCR amplification was performed using primers with T7 promoter. VgR and GFP dsRNA were synthesized with T7 RiboMAX^TM Express RNAi System synthesis kit. The abdomens of S. exigua female pupae on 2 nd and 6 th day were injected with 3 μL double RNA by 10 μL microsyringe(2 μg·μL^-1). RT-qPCR was used to detect the changes of VgR expression in 0-, 24-, 48-hour-old female adults. Meanwhile, eclosion rate and eggs per female were evaluated in control groups(blank control, GFP-ds RNA injection) and treatment group(VgR-dsRNA injection). 【Result】 The VgR and GFP gene fragments obtained by amplification were 327 and 417 bp, respectively. The VgR expression level of 0-, 24-, 48-hour-old female adults in the VgR-dsRNA group decreased by 79.35%, 84.22% and 67.68% compared with the GFP-dsRNA group, respectively. Through anatomical observation of the ovary of 0-, 24-, 48-hour-old female adults, it was found that compared with the GFP-ds RNA group, the ovary development of the VgR-dsRNA group was significantly delayed. Compared with the GFP-ds RNA group, the length of the ovary tube in the VgR-dsRNA group decreased by 2

关 键 词：甜菜夜蛾 卵黄原蛋白受体基因 RNA干扰 卵巢发育 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]