四种鳞翅目害虫精氨酸激酶基因的表达谱及基于RNAi的功能分析

Expression profiling and RNAi-based function analysis of arginine kinase gene in four lepidopteran pests

【目的】精氨酸激酶(arginine kinase,AK)(EC2.7.3.3)是昆虫体内重要的磷酸原激酶(能量代谢调节因子),也是唯一能够形成有效ATP的磷酰基供体,起着与脊椎动物中肌酸激酶相同的作用。本研究旨在了解鳞翅目害虫AK基因的表达和功能。【方法】利用qRT-PCR方法测定AK基因在大螟Sesamia inferens、二化螟Chilo suppressalis、甜菜夜蛾Spodoptera exigua和斜纹夜蛾Spodoptera litura这4种鳞翅目害虫不同发育阶段和3龄幼虫不同组织中的表达谱;通过终点法检测了这4种害虫不同发育阶段和幼虫不同组织中的AK酶活性;采用RNAi技术抑制该基因的表达并分析其功能。【结果】AK基因在大螟、二化螟、甜菜夜蛾和斜纹夜蛾这4种鳞翅目昆虫的不同发育阶段和3龄幼虫不同组织中均有表达,说明该基因的表达不具有发育时期和组织特异性。不同发育时期和3龄幼虫不同组织中AK酶活性与基因表达量变化趋势大体一致。注射以AK基因为靶标的dsRNA6d后,4种害虫体内AK基因的mRNA表达下降30%~50%,AK酶活性降低30%左右;14d后幼虫的死亡率达50%左右,显著高于对照组幼虫的死亡率。【结论】AK基因在上述4种鳞翅目害虫中为组成型表达,RNAi抑制AK基因的表达可导致4种害虫的幼虫死亡,研究结果为开发以AK基因为靶标的鳞翅目害虫防治新技术提供了理论依据。

【Aim】 Arginine kinase (AK) is an important phosphogen kinase (regulation factor of energy metabolism) in insects and the only phosphoryl donor that can form effective ATP.It plays the same role as creatine kinase in vertebrates.This study aims to clarify the expression and function of AK gene in lepidopteran pests.【Methods】 We determined the expression profiles of AK gene in different developmental stages and tissues of the 3rd instar larvae by qRT-PCR,and assayed the AK activity in four lepidopteran pests,Sesamia inferens,Chilo suppressalis,Spodoptera exigua and Spodoptera litura by endpoint method.Furthermore,we analyzed the biological functions of AK genes in these four species by RNAi.【Results】 qRT-PCR assay revealed that AK gene was expressed at all the developmental stages and in various tissues of the 3rd instar larvae of these four species,indicating that the expression of AK gene has no developmental stage and tissue specificity.The change trend of AK activities in different developmental stages and tissues of the 3rd instar larvae were similar with that of the expression levels of AK gene.At 6 d post injection of dsRNA targeting AK gene,the expression level of AK gene decreased by 30%-50% and the AK activity decreased by about 30% in these four species.At 14 d post injection of dsRNA targeting AK gene,the larval mortality rates of the four species were around 50%,which was significantly higher than those of the control groups.【Conclusion】 AK gene is constitutively expressed in the four lepidopteran pests.Inhibition of AK gene expression by RNAi can lead to the larval death of these four species.Our results provide a theoretical basis for developing new lepidopteran pest control technologies targeting AK gene.