RNAi技术介导舞毒蛾热激蛋白Hsp40基因功能分析

摘要 [目的]测定Hsp40基因沉默对舞毒蛾生长发育及Hsp40基因表达量的影响。[方法]体外合成双链RNA（dsRNA），并将dsRNA通过微注射入舞毒蛾3龄幼虫体内，测定Hsp40基因沉默对舞毒蛾生长发育及Hsp40基因表达量的影响。[结果]分别注射ddH₂O、dsRNAGFP和dsRNAHsp40 后8 d，dsRNAGFP处理组舞毒蛾幼虫相对取食量显著高于ddH₂O和dsRNAHsp40处理组（P<0.05）；但3种处理对舞毒蛾幼虫的相对生长率、食物利用率、食物转化率、近似消化率方面均无显著性差异。注射后4 d，ddH₂O处理组的舞毒蛾幼虫体重累计增长率最大，其次是dsRNAHsp40处理组，dsRNAGFP处理组的体重累计增长率最小，这与4 d的幼虫鲜重一致；其余时间点，dsRNAHsp40处理组的体重累计增长率均大于ddH₂O和dsRNAGFP处理组。将1 μl（1 μg/μl）的dsRNA注射入舞毒蛾幼虫体内，6～48 h Hsp40基因表达量显著下降，96 h基因表达量上调。[结论] 该研究为进一步利用沉默舞毒蛾Hsp40基因在害虫防治中的应用提供理论依据。

关键词 舞毒蛾；Hsp40；RNA干扰；基因沉默

中图分类号 S188 文献标识码

A 文章编号 0517-6611（2014）26-08890-04

Functional Analysis of Hsp40 Gene in Lymantria dispar Mediated RNAi Technology

WANG Zhi-ying et al （College of Forestry， Northeast Forestry University， Harbin，Heilongjiang 150040）

Abstract [Objective] The aim was to measure the effects of Hsp40 gene silencing on growth and Hsp40 gene expression of L.dispar larvae.[Method] The 1 μg/μl of double-stranded RNA （dsRNA） in vitro synthesized，was microinjected into 3rd instar Lymantria dispar larvae. The effects of Hsp40 gene silencing on growth and Hsp40 gene expression of L. dispar larvae were measured. [Result]The results showed the relative consumption rate （RCR） of L. dispar larvae microinjected by dsRNAGFP was higher than those microinjected by ddH₂O and dsRNAHsp40 at 8 d time point. However， relative growth ratio （RGR）， efficiency of conversion of ingested food （ECI）， approximate digestibility （AD）， efficiency of conversion of digested food （ECD） of L. dispar larvae among three treatments were no significant differences. After 4 d of microinjection， the weight cumulative growth rates of L. dispar larvae were decreasing order of ddH₂O， dsRNAHsp40 and dsRNAGFP， which were consistent with the larvae weight. At the rest of time points， weight cumulative growth rate of dsRNAHsp40 treatment group were greater than other treatment groups. After microinjection 1 μg/μl of dsRNA into the L. dispar larvae ranged from 6 h to 48 h， Hsp40 gene expressions were significantly decreased，while those increased at 96 h. [Conclusion]These results provided a theoretical basis for further silencing Hsp40 gene of L. dispar into pest control.

Key words Lymantria dispar；Hsp40；RNAi；Gene silencing

NAPOLI等^[1]将外源基因引入矮牵牛（Petunia hyhrida Vilm）后发现内源同源基因出现沉默，将此现象称为共抑制。GUO等^[2]用反义RNA阻断线虫基因表达的试验中发现，反义和正义RNA都阻断了基因的表达。1998年，FIRE等^[3]在线虫 （Caenorhabditis elegans）试验中发现在体外转录正义RNA时生成的双链RNA（double strand RNA，dsRNA）导致基因表达的阻断。将由dsRNA引发生物体同源序列mRNA降解而最终导致特异性基因转录后沉默的效应称为RNA干扰（RNA interference，RNAi）。这一现象主要是通过dsRNA被一种称为Dicer的核酸酶切成21～25 nt的干扰性小RNA片段（siRNA），由siRNA介导识别并靶向切割同源性靶mRNA分子而实现基因沉默^[4-7]。现已在真菌、植物、线虫、昆虫、哺乳动物等许多真核生物体内都发现了这种现象。由于RNAi诱导基因沉默的特异性^[8]和高效性，特别是在非模式生物中操作的简便性，被广泛应用于多种生物的基因功能研究和有害生物控制研究^[9-10]，并实现了对鳞翅目昆虫烟草天蛾（Manduca sexta）^[11-12]、斜纹夜蛾（Spodoptera litura）^[13]和棉铃虫（Helicoverpa armigera）^[14]等虫体内部分基因功能的研究。