利用RNAi技术沉默麦长管蚜与桃蚜细胞色素P450

【目的】通过体外饲喂双链RNA（dsRNA），利用RNAi技术沉默麦长管蚜和桃蚜体内细胞色素P450。【方法】采用PCR克隆获得麦长管蚜和桃蚜体内细胞色素P450片段；利用双链RNA合成试剂盒合成麦长管蚜和桃蚜细胞色素P450保守序列的dsRNA；在蚜虫人工饲料中，分别加入终浓度为0、3、5和7.5 ng?μL-1的dsRNA，每个试验组设置3个重复，分别于饲喂后2天、4天、6天、8天统计蚜虫的存活数；利用实时荧光定量PCR技术分析饲喂后目标基因的表达抑制情况。【结果】从麦长管蚜与桃蚜的cDNA中分别克隆细胞色素P450，所克隆片段同源性为90.1%，发现随着dsRNA浓度的增大，麦长管蚜和桃蚜的死亡率随着时间的推移不断升高，最高死亡率可分别达75.56%和64.44%。提取饲喂dsRNA浓度为7.5 ng?μL-1的不同时间段的蚜虫总RNA，实时荧光定量PCR结果表明，目的基因被沉默。【结论】利用麦长管蚜和桃蚜细胞色素P450保守序列的dsRNA，体外饲喂后，通过RNA干扰（RNAi），能够显著降低蚜虫体内细胞色素P450的表达水平，直至导致目的基因沉默和蚜虫死亡。

Silencing of Cytochrome P450 in Sitobion avenae and Myzus persicae Through RNA Interference

【Objective】Silencing of cytochrome P450 in S. avenae and M. persicae through RNA interference by feeding on artificial diet containing double-stranded RNA (dsRNA).【Method】 Fragments of cytochrome P450 in S. avenae and M. persicae were obtained by PCR. The dsRNA of the conserved sequence of cytochrome P450 from S. avenae and M. persicae were synthesized by dsRNA synthesis kit. dsRNA was added to the final concentration of 0, 3, 5, and 7.5 ng?μL-1 in aphid artificial diet, respectively. Three replicates were set up for each experimental group. The number of surviving aphids was counted after 2, 4, 6, and 8 days of feeding. The expression level of target genes were analyzed by real-time fluorescence quantitative PCR.【Result】Cytochrome P450 fragments were cloned from the cDNA of S. avenae and M. persicae, the homology of the two genes was 90.1%. The mortality of aphids increased along with the increased dsRNA concentration as time went on. Total RNA were extracted from aphids fed by dsRNA at concentration of 7.5 ng?μL-1 and collected from different time periods. The results of real-time fluorescence quantitative PCR showed that the target genes were silenced significantly. 【Conclusion】The expression level of cytochrome P450 in S. avenae and M. persicae which fed by the dsRNA of the conserved sequences of cytochrome P450, were suppressed, and thus led to the death of aphids.