褐飞虱海藻糖酶基因在表皮几丁质代谢中的调控作用

【目的】昆虫海藻糖酶能够调控几丁质代谢并控制蜕皮过程。本研究通过TRE表达被抑制后,检测褐飞虱(Nilaparvata lugens)蜕皮状况、几丁质含量及几丁质合成酶(chitin synthase,CHS)和几丁质酶(chitinase,Cht)基因表达情况,探究不同的海藻糖酶(trehalase,TRE)在褐飞虱表皮中对几丁质代谢的调控作用。【方法】采用RNAi技术,以实验室饲养种群褐飞虱为材料,通过向其体内注射双链RNA(dsRNA)分别抑制单个海藻糖酶基因或同时抑制多个海藻糖酶基因,注射48 h后通过Trizol法提取褐飞虱总RNA,反转录试剂盒合成第一链DNA后采用实时荧光定量PCR(qRT-PCR)技术检测该基因的表达情况,确定RNAi效果。氢氧化钾法测定48 h褐飞虱整体几丁质含量变化并对蜕皮困难虫体进行拍照;最后采用qRT-PCR检测褐飞虱CHS和Cht在mRNA水平上的相对表达量变化,分析TRE在调控几丁质代谢中的作用。【结果】与注射dsGFP相比较,其余各注射组褐飞虱整体几丁质含量显著下降,其中dsTRE1混合注射组与Validamycin注射组呈极显著下降,同时褐飞虱出现蜕皮困难等现象。qRT-PCR检测结果显示单个TRE的dsRNA注射后该基因的表达被抑制,但是部分TRE的表达有互补性上升。其中TRE1-2和TRE2在各注射组处理下表达均下降,dsTRE1s对TRE2的表达也有抑制效果,整体上dsTRE1混合注射组和海藻糖酶抑制剂Validamycin抑制效果明显;dsTRE注射组抑制CHS表达效果不明显,Validamycin能够显著降低CHS1和CHS1a在表皮中的表达,且2种dsTRE1注射后CHS1表达在上升,dsTRE1-2注射后表皮中的CHS1a的表达上升;Cht1和Cht8在dsTRE各注射组及Validamycin处理中表达下降或显著下降,dsTRE1-1注射后Cht2和Cht5表达显著上升;dsTRE1-2注射后Cht1、Cht6和Cht8表达下降,Cht2和Cht4表达显著上升;dsTRE2处理组中Cht1、Cht8和Cht10表达下降而Cht9表达显著上升;dsTRE1s注射后,Cht1和Cht5表达显著下降,而Cht9表达显著上升;Validamycin注射组中10个几丁质酶基因表达都显著或者极显著下降。【结论】TRE能够通过调控褐飞虱几丁质代谢途径来控制几丁质的合成,结果可为开展和筛选有效的海藻糖酶抑制剂控制褐飞虱等害虫提供理论依据。

Regulatory Function of Trehalase Genes on Chitin Metabolism in the Cuticle of Nilaparvata lugens

【Objective】The previous research results showed that insect trehalase (TRE) can regulate chitin metabolism and control the molting process. In this study, the brown planthopper (Nilaparvata lugens) molting process, the changes of the expression of chitin content and chitin synthase (CHS) and chtinase (Cht) genes were detected when TRE genes were knocked down by the way of RNAi, in order to explore the roles of different trehalase genes in the regulation of chitin metabolism in the epidermis.【Method】N. lugens fed in the lab was chosen as the experimental material, and RNAi technology was used to inhibit the single or two TRE genes’ expression by injection of double stranded RNA. The total RNA was extracted from the cuticle of N. lugens using the TRIzol^® reagent as instructed by manufacturer. First-stand cDNA was synthesized using the PrimeScript^TM RT reagent Kit with gDNA Eraser following the manufacturer’s instructions. And the effect of RNAi was firstly determined after 48 h of injection by quantitative real-time PCR (qRT-PCR). Secondly, the chitin content of N. lugens whole body was determined at 48 h using potassium hydroxide method qRT-PCR, and photos of the insects with molting difficulties were taken in the same time. In the last, the relative expression levels of CHS and Cht of N. lugens were detected by qRT-PCR, and the regulatory function on chitin metabolism of TRE was analyzed at the same time. 【Result】Compared with the injection of dsGFP which was used as a control group, the results showed that in other groups injected with dsRNA the chitin content of N. lugens was decreased significantly, in which the dsTRE1 mixed injection group and the Validamycin injection group showed a significant decrease, meanwhile its molting problems appeared at the same time. qRT-PCR results showed that the gene expression of individual TRE was inhibited at 48 h after one TRE dsRNA injection, and the other TRE expression was increased and indicated it has a complementary function. The expressions of TRE1-2 and TRE2 were decreased in all groups, and dsTRE1s also inhibited the expression of TRE2. Secondly, the obvious effects could be found when mixed dsTRE1 trehalase inhibitor Validamycin injected into N. lugens and TRE genes were decreased significantly. The expression level of CHS and its splicing variants had no obvious effect when every TRE genes’ expression was knocked down, while CHS1 and CHS1a expressions were significantly decreased at 48 h after Validamycin injection. The expression of CHS1 in the cuticle increased after dsTRE1-2 injection and the expression of CHS1a increased after injection of dsTRE1-2. Thirdly, the expression levels of Cht1 and Cht8 decreased or decreased significantly after four dsTRE and Validamycin injection. The expression levels of Cht2 and Cht5 increased significantly when dsTRE1 was injected, as well as Cht2 and Cht4 increased significantly while Cht1, Cht6 and Cht8 decreased after dsTRE1-2 injection and Cht2 expressed increased significantly while the expression of Cht1, Cht8 and Cht10 decreased at 48 h when TRE2 knocked down. In the same time, the expressions of Cht1 and Cht5 decreased significantly while Cht9 increased significantly at 48 h after dsTRE1s injection. In the last, about all of 10 chitinase genes’ expression decreased significantly or extremely significantly after Validamycin injection.【Conclusion】TRE can control the synthesis of chitin through the regulation of chitin metabolic pathways in N. lugens. The results of this study will provide a theoretical basis for developing and screening effective trehalase inhibitors to control N. lugens.