东亚三角涡虫DjStag基因干扰载体的构建及干扰效果鉴定

Construction of Interference Vector of planaian(Dugesia japonica) DjStag and Identification of Its Interference Efficiency

构建DjStag基因的RNA干扰表达载体,诱导表达产生dsRNA后喂食涡虫,观察DjStag表达变化及对涡虫再生的影响。本文以含有东亚三角涡虫DjStag基因的pcDNA3-DjStag重组质粒为模板,经PCR扩增目的片段,将其克隆到干扰载体L4440上,构建重组质粒L4440-DjStag后转化入大肠杆菌HT115感受态细胞中,IPTG诱导表达dsRNA后喂食涡虫。显微观察涡虫再生过程中的表型变化,Real-time PCR检测干扰后涡虫DjStag基因表达的变化。结果表明,成功构建了DjStag基因的RNA干扰表达载体;DjStag基因RNA干扰后,涡虫不能正常再生,再生片段显示出明显的再生缺陷;涡虫体内DjStag基因的表达受到抑制,DjStag mRNA的表达水平显著下降。

The objective of this study was to construct the RNA interference vector expressing DjStagdsRNA and investigate the effect of dsRNA on regeneration in planarians Dugesia japonica. In the present study, the cDNA fragment for the DjStag from adult planarians Dugesia japonica cDNA library wasamplified from recombinant plasmid pcDNA3-DjStag using PCR and then cloned into the interference vector 1.4440. The recombinant plasmid L4440-DjStag was transferred into E. coli HTll5 to makedsRNA induced by IPTG. RNA interference （RNAi） food is fed directly to planarians then DjStag RNAi animals were cut head and tail and allowed to regenerate. Animals were observed with a stereo-microscope during the process of regeneration. The interference efficiency was assessed using the Relative quantitative real-time PCR method. Results showed that the RNA interference vector IA440-DjStagwas successfully constructed. Regeneration was severely impaired and the expression of DjStag mRNA was significantly decreased after RNAi compared to normal regenerating planarians. The RNAi inhibitory effect on the regeneration of planarians is confirmed.