摘要
RNA干扰(RNA interference,RNAi)是一种调控基因表达的方法,其通过体外合成一段与内源靶基因同源的双链RNA(dsRNA)或siRNA,导入生物体内,使内源靶基因中同源mRNA降解,从而达到阻抑基因表达的目的。类钙粘蛋白(cadherin-likeprotein)是位于昆虫中肠刷状缘膜囊(brush border membrane vesicles,BBMV)上与钙粘蛋白(cadherin)结构相似的物质,是多种昆虫体内Bt杀虫蛋白的受体。本研究利用基因特异引物通过RT-PCR扩增了小菜蛾类钙粘蛋白基因的2个片段(CAD1和CAD2),合成相对应的双链RNA(double-stranded RNA,dsRNA);并将dsRNA通过显微注射导入小菜蛾3龄幼虫体内,测定了不同靶位点、不同剂量、不同检测时间对目的基因mRNA表达量的影响。结果表明:将70nLCAD1对应的dsRNA注射到幼虫体内48h后,基因表达量显著下降,72h后恢复。免疫印迹检测结果表明,类钙粘蛋白在注射dsRNA48h后幼虫BBMV中的含量明显下降。本实验成功实现了小菜蛾类钙粘蛋白基因的沉默,该体系的成功建立为利用RNAi技术分析小菜蛾及其他鳞翅目昆虫基因的功能提供了参考。
RNA interference （RNAi） is the process of sequence-specific post-transcriptional gene silencing initiated by double-stranded RNA （dsRNA） which is homologous in sequence to the silenced gene. The cadherin-like protein, similar to cadherin, lies in brush border membrane vesicles （BBMV） of insect. It had been confirmed to be a receptor of Bacillus thuringiensis （Bt） insecticidal protein in several insects. In this study, two cDNA segments （CAD1 and CAD2） encoding the cadherin-like protein gene were cloned through RT-PCR technique using gene specific primers; double-stranded RNA （dsRNA） was synthesized by using the two gene segments respectively, and microinjected into 3rd instar larvae of Plutella xylostella ; and the effects of different target sites, dose and silencing time on cadherin mRNA expression level were assayed. The results showed that the expression of cadherin-like gene was reduced sharply at 48 h after the dsRNA corresponding to CAD1 segment was injected and restored at 72 h. The immunoblotting results indicated that cadherin-like protein in BBMV of P. xylostella was reduced markedly at 48 h after injection. The cadherin-like gene was silenced successfully in this experiment, and the establishment of RNAi technique can help reveal the gene function of P. xylostella including cadherin-like gene.
出处
《昆虫学报》
CAS
CSCD
北大核心
2009年第8期832-837,共6页
Acta Entomologica Sinica
基金
"973"计划项目(2006CB102000)
国家自然科学基金项目(30871659)
"863"计划项目(2007AA10Z421)
农业部行业项目(2008326001)
