镰形扇头蜱Subolesin基因的长链dsRNA电转化和干扰效果研究  

The electroporation of long chain dsRNA targeting Subolesin gene and effect of RNA interference in Rhipicephalus haemaphysaloides

作　　者：路鹏云[1,2] 龚海燕[2] 曹杰[2] 周勇志[2] 张厚双[2] 李国清[1] 周金林[2] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国预防兽医学报》2015年第4期274-278,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31172095)

摘　　要：为建立针对不同发育阶段蜱的长链dsRNA电转化导入方法,本研究以我国优势流行的硬蜱镰形扇头蜱为对象,以蜱的保守基因Subolesin作为目标基因,将荧光标记的Subolesin-dsRNA对卵、幼蜱以及若蜱3个发育阶段进行电转化监测dsRNA的转化效果,对电转化后不同时间以及不同dsRNA电转化浓度进行优化,并接种小鼠观察电转化Subolesin-dsRNA对幼蜱及若蜱吸血的影响。结果显示,电转化Cy3荧光染料标记dsRNA后,卵、幼蜱及若蜱体内均有点状分布的荧光。若蜱于电转化后放置8h,Subolesin mRNA沉默效果较佳。此外,卵、幼蜱及若蜱分别在dsRNA浓度为10μg/m L、5μg/m L^25μg/mL及10μg/m L^25μg/m L沉默效果较好。Subolesin-dsRNA干扰后蜱的存活率和吸血能力均显著降低。本研究建立的针对各个发育阶段蜱的高效长链dsRNA电转化方法为研究基因功能和沉默特定的基因提供了新的ds RNA导入方法。In order to optimize the method to deliver the long chain of dsRNA into Rhipicephalus haemaphysaloides at different life stages by electroporation, the fluorescence-labeled dsRNA targeting the conserved sequence of Subolesin gene was transduced by electroporation into eggs, larva and nymph of the tick, respectively, which is widely prevalence in China. And the optimization included the concentration and optimum effective time after electroporation. In addition, the knock-down effect of the RNA interference （RNAi） on Subolesin gene expression in larva and nymphs was investigated after a blood-feeding on mice. As a result, the specific fluorescence as the indicator from the Cys3-1abeled dsRNA was detected in the body of larva and nymphs. The highest transduction rate in ticks was achieved at 8 hours post-electroporation. And the optimized concentration of dsRNAs was 10μg/mL, 5 to 25 μg/mL and 10 to 25 μg/mL used for the electroporation in eggs, larva and nymphs, respectively. RNAi knock- down of Subolesin expression resulted in a significant decrease of the survival rate and feeding ability of the ticks. The optimized electroporation method in the present study provided a basis for silencing the gene expression in ticks.

关 键 词：电转化 RNA干扰 DSRNA 荧光标记 

分 类 号：S852.7[农业科学—基础兽医学]