白背飞虱海藻糖合成酶基因调控几丁质合成的功能

Regulation Function of Trehalose-6-phosphate Synthase Genes on Chitin Synthesis in Sogatella furcifera

【背景】海藻糖合成酶(trehalose-6-phosphate synthase,TPS)在海藻糖合成中起着重要作用,其能够介导海藻糖代谢调控几丁质合成及昆虫发育。【目的】本研究通过抑制白背飞虱(Sogatella furcifera)TPS的表达,检测RNAi沉默SfTPS效果,观察白背飞虱蜕皮状况,测定几丁质含量及几丁质合成酶(chitin synthase,CHS)基因的定量表达,探究SfTPS在白背飞虱几丁质合成中的潜在调控作用。【方法】利用注射法RNAi技术,以实验室饲养多年的白背飞虱种群为试验材料,体外合成两个SfTPS(SfTPS1和SfTPS2)与GFP的双链RNA(dsRNA)后,分别注射到白背飞虱体内抑制TPS。首先,在dsRNA注射后48 h采用Trizol法提取白背飞虱的总RNA,反转录并合成第一链DNA后,采用实时荧光定量PCR(qRT-PCR)技术检测TPS表达沉默情况,以确定RNAi的效果;其次,测定dsRNA注射后48 h和72 h白背飞虱整体几丁质含量并对翅发育畸形虫体进行拍照;最后,采用qRT-PCR技术检测白背飞虱SfCHS在mRNA水平上的相对表达量变化,分析SfTPS1和SfTPS2在几丁质合成调控中的作用。【结果】与注射dsGFP相比较,dsSfTPS1和dsSfTPS2的RNA注射后,能够促进SfCHS表达量上升,几丁质含量增加,白背飞虱成虫翅出现畸形。qRT-PCR结果显示,单个SfTPS dsRNA注射后本基因的表达能够被极显著抑制,与注射dsGFP相比,不足对照组表达量的30%,且单个SfTPS的dsRNA注射后,另外一个SfTPS表达同样显著下降;dsSfTPS1和dsSfTPS2注射后,白背飞虱成虫翅均为长翅,出现一定比例的翅卷曲等畸形情况,其后48 h和72 h产生一定的死亡率;几丁质含量检测发现,SfTPS1和SfTPS2的dsRNA注射后72 h,几丁质含量显著上升。与注射dsGFP对照组相比较,SfCHS1与SfCHS1a表达量在dsSfTPS1注射后72 h极显著上升,在dsSfTPS2注射后48 h和72 h时极显著上升,且dsSfTPS1和dsSfTPS2注射后SfCHS1b的表达极显著增加。【结论】SfTPS能够通过调控白背飞虱几丁质合成酶基因的表达来控制几丁质的合成,研究结果有助于评价SfTPS在白背飞虱等昆虫中的调控作用并作为潜在控害靶标,为进一步开展和筛选有效的海藻糖合成酶抑制剂控制白背飞虱等害虫提供理论依据。

【Background】It is well known that trehalose-6-phosphate synthase(TPS)plays an important role in trehalose synthesis,which can mediate trehalose metabolism to regulate chitin synthesis and insect development.【Objective】In this study,the effect of silencing SfTPS was detected,the molting status of Sogatella furcifera was observed and the content of chitin and the quantitative expression of chitin synthase(CHS)gene were determined through inhibiting the expression of TPS by RNAi in S.furcifera.The purpose is to explore the potential regulatory effects of SfTPS on the chitin synthesis in S.furcifera.【Method】The S.furcifera population,which has been fed in laboratory for many years,was used as experimental material.The double-stranded RNA(dsRNA)of SfTPS and GFP were synthesized in vitro in order to study the potential function of TPSs,the RNA of two dsSfTPSs(dsSfTPS1 and dsSfTPS2)was injected into S.furcifera,respectively.Firstly,after dsRNA injection,the total RNA of S.furcifera was extracted by Trizol method at 48 h,and the first strand DNA was synthesized as the template of quantitative real-time PCR(q RT-PCR).As well as qRT-PCR was used to detect the silencing of SfTPS expression for the effect of RNAi.Secondly,the whole chitin of S.furcifera was detected at 48 and 72 h after dsRNA injection,and photographs were taken of the winged developmental malformations.Finally,the relative expression level of SfCHS in S.furcifera was detected by qRT-PCR,and the roles of SfTPS1 and SfTPS2 in the regulation of chitin synthesis were analyzed.【Result】Compared with dsGFP injection,the expression of SfCHS increased after dsSfTPS1 and dsSfTPS2 injection,as well as the chitin content increased,which lead to the wing deformity of S.furcifera.The q RT-PCR results showed that the expression of SfTPS was significantly inhibited after dsRNA injection of a single SfTPS,which was less than 30%of that in the control group injected with dsGFP.In addition,the expression of another SfTPS also decreased significantly after a single SfTPS dsRNA injection.The adult wings of S.furcifera were all long wings after dsSfTPS1 and dsSfTPS2 injection.Some deformities such as wing curl in a certain ratio had been found in these long wings,and a certain ratio of mortality was found at 48 and 72 h.The chitin content increased significantly at 72 h after these two dsSfTPSs RNA injection.Compared with the control group,the expression of SfCHS1 and SfCHS1 a increased significantly at 72 h after dsSfTPS1 injection,followed it increased significantly at 48 and 72 h after dsSfTPS2 injection.In the same time,the expression of SfCHS1 b increased significantly after dsSfTPS1 and dsSfTPS2 injection.【Conclusion】The SfTPS can control the synthesis of chitin through the regulation of chitin synthase gene in S.furcifera.The results are helpful to evaluate the regulatory role of SfTPS in S.furcifera and other insects and as the potential pest control target.It provides a theoretical basis for further development and screening of effective trehalose-6-phosphate synthase inhibitors to control S.furcifera pests and so on.