摘要
目的 近年来 ,双链RNA介导的基因沉默 (RNA干扰 )已在许多动植物中发现 ,并成为研究基因功能的强有力的工具。为确定蚊虫细胞中是否有RNA干扰现象 ,以期为利用RNAi效应消除蚊虫抗药性研究奠定基础。 方法 设计 5′端带T7启动子的绿色荧光蛋白 (GFP)及糜蛋白酶特异性引物 ,PCR扩增目的基因获得 5′端带有T7启动子DNA片段 ,体外转录成单链RNA ,退火后形成双链RNA。转染带有GFP基因的昆虫细胞表达载体 pAct .GFP和双链RNA进入C6/ 3 6蚊虫细胞 ,72h后收集细胞 ,用激光共聚焦显微镜和流式细胞仪检测GFP的表达差异 ,用半定量RT PCR检测GFP的mRNA的表达水平。 结果 转染糜蛋白酶基因双链RNA和 pAct .GFP质粒的细胞及仅转染pAct .GFP的细胞的GFP表达水平没有明显差异 ;转染GFP基因的双链RNA和pAct .GFP质粒的细胞的GFP表达水平显著下降 ,达 40 %～ 5 0 % ,GFP的mRNA的表达水平也相应下降了 60 %。 结论在蚊虫细胞内 ,双链RNA能特异性的抑制相应的同源性基因的表达 ,引起基因沉默 。
Objective Recently, a naturally occurring gene silencing mechanism triggered by double-stranded RNA(dsRNA), RNA interference(RNAi), has been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells. Methods Individual DNA fragments coding for 690 bp GFP gene were amplified in separate PCR using pEGFP-C3 plasmid DNA as a template and primer pairs with minimal T7 promoter sequence appended to the 5′ end of the primers, and then transcript dsRNA in vitro. C6/36 Plasmid and/or dsRNA were transfected to C6/36 mosquito cells. GFP protein expression was assessed using confocal laser microscope and flow cytometer, and the expression level of GFP mRNA was determined by relative quantitative RT-PCR. Results The cotransfection of pAct.GFP and the control chymotrypsin dsRNA and only the transfection of pAct.GFP gave the similar percentage of cell expressing GFP, but the GFP dsRNA significantly reduced both the percentage and fluorescence intensity of GFP positive cells by approximately 40%-50%, and 60% of reduction in the levels of GFP mRNA. Conclusion This study demonstrates dsRNA-triggered inhibition of gene expression and sequence-specific inhibition in mosquito cells.
出处
《中国寄生虫病防治杂志》
CSCD
2004年第5期261-263,F003,共4页
Chinese Journal of Parasitic Disease Control
基金
国家自然科学基金资助项目 (No.30 371 755)
南京医科大学创新基金项目 (No .CX 2 0 0 2 0 0 7)~~
关键词
白纹伊蚊
RNA干扰
双连RNA
基因沉默
C6/36细胞
Aedes albopictus
RNA interference(RNAi)
double-stranded RNA(dsRNA)
gene silencing
C6/36 cell
