抑咽侧体素基因在大猿叶虫生殖滞育准备中的功能分析

Analysis of the functions of allatostatin genes in the reproductive diapause preparation of Colaphellus bowringi(Coleoptera:Chrysomelidae)

【目的】本研究旨在明确大猿叶虫Colaphellus bowringi促咽侧体素(allatotropin,AT)和抑咽侧体素(allatostatin,AST)基因的分子特征,分析其在该虫生殖和滞育过程中的表达差异,并探究其在大猿叶虫生殖滞育准备中的功能。【方法】利用前期建立的大猿叶虫转录组数据库,鉴定大猿叶虫CbAT和CbAST s基因序列并克隆其开放阅读框(ORFs),并对其序列进行生物信息学分析;通过qRT-PCR技术检测CbAT和CbAST s在大猿叶虫注定滞育和注定非滞育雌蛹和雌成虫头部的表达模式;对注定滞育大猿叶虫2日龄雌蛹进行CbAST-B,CbAST-C以及CbAST-B+CbAST-C RNAi试验后,检测4日龄雌成虫保幼激素(juvenile hormone,JH)信号基因以及卵黄原蛋白基因的表达量变化。【结果】鉴定并克隆了大猿叶虫的一个促咽侧体素基因CbAT(GenBank登录号:MT977128)及两个抑咽侧体素基因CbAST-B(GenBank登录号:MT977126)和CbAST-C(GenBank登录号:MT977127),其开放阅读框分别长408,600和303 bp。通过氨基酸序列比对和系统进化分析发现,CbAT,CbAST-B和CbAST-C均与鞘翅目昆虫的同源蛋白聚在一支,在进化上表现出较高保守性。qRT-PCR检测发现,CbAT基因在注定滞育与注定非滞育2日龄雌蛹到4日龄雌成虫头部中始终无表达差异;而CbAST-B和CbAST-C基因分别自雌成虫1和2日龄开始在注定滞育雌成虫头部显著高表达,且在注定滞育和注定非滞育个体中的差异表达状态一直持续到滞育准备期结束。在注定滞育2日龄雌蛹中沉默CbAST-B,CbAST-C以及同时沉默CbAST-B和CbAST-C后,检测4日龄雌成虫头部的干扰效率发现,CbAST-B和CbAST-C的表达均受到显著抑制,干扰效率达70%以上。在去除触角的头部中,JH合成途径基因AACT,FPPS和JHAMT以及脂肪体中JH应答基因Kr-h1和JHE1的表达在ds CbAST-B,ds CbAST-C和ds CbAST-B+ds CbAST-C处理组中均被显著上调,脂肪体中卵黄原蛋白基因Vg1和Vg2的表达也被显著上调。【结论】CbAT基因可能并非造成大猿叶虫产卵前期与滞育准备期保幼激素信号产生差异的主要原因,而在生殖滞育准备期CbAST-B和CbAST-C抑制了JH的合成,进而抑制Vg s的表达以促进滞育的发生。本研究进一步揭示了昆虫生殖滞育准备期保幼激素信号的上游调控机制,有助于进一步理解昆虫对环境的季节性适应策略,为挖掘害虫防治的新靶标提供了新的指导和借鉴。

【Aim】This study aims to clarify the molecular characteristics and expression difference of allatotropin(AT)and allatostatin(AST)genes during the reproduction and diapause of Colaphellus bowringi,so as to further reveal the functions of these genes in the reproductive diapause preparation of C.bowringi.【Methods】We identified the sequences of CbAT and CbAST s in C.bowringi based on the previously constructed transcriptome database,cloned their ORFs and carried out the bioinformatics analysis of their sequences.Then,we assayed the expression patterns of CbAT and CbAST s in the heads of the diapause-destined and non-diapause-destined female pupae and female adults via qRT-PCR.RNAi experiments of CbAST-B,CbAST-C and CbAST-B+CbAST-C were done in the diapause-destined 2-day-old female pupae,and the expression levels of juvenile hormone(JH)signaling genes and vitellogenin genes in 4-day-old female adults were detected.【Results】One allatotropin gene CbAT(GenBank accession no.:MT977128)and two allatostatin genes CbAST-B(GenBank accession no.:MT977126)and CbAST-C(GenBank accession no.:MT977127)of C.bowringi were identified and cloned,with the ORFs of 408,600 and 303 bp,respectively.By amino acid sequence alignment and phylogenetic analysis,we found that CbAT,CbAST-B and CbAST-C respectively cluster into the same branch with homologous proteins of Coleoptera insects,showing high conservation in the evolution.The qRT-PCR assay indicated that CbAT had no significant expression differences in the female heads between the diapause-destined and non-diapause-destined individuals from the 2-day-old pupal stage to the 4-day-old adult stage.CbAST-B and CbAST-C were significantly highly expressed in the heads of diapause-destined female adult since the 1-and 2-day-old,respectively,and the differential expression state between the diapause-destined and non-diapause-destined individuals lasted until the end of diapause preparation period.After knocking down CbAST-B,CbAST-C and CbAST-B+CbAST-C in the diapause-destined 2-day-old female pupae,the expression of CbAST-B and CbAST-C in the heads of 4-day-old female adults was significantly suppressed with the RNAi efficiency over 70%.Further study revealed that the expression of JH biosynthesis genes AACT,FPPS and JHAMT,in the head without antenna,JH-responsive genes Kr-h1 and JHE1 in the fat body,and vitellogenin genes Vg1 and Vg2 in the fat body was all significantly up-regulated in the treatment groups of ds CbAST-B,ds CbAST-C and ds CbAST-B+ds CbAST-C.【Conclusion】CbAT may be not a key regulator mediating the differences of JH signaling between the preoviposition period and diapause preparation phase in C.bowringi.However,CbAST-B and CbAST-C suppress the synthesis of JH and further down-regulate the expression of Vg genes in C.bowringi during the reproductive diapause preparation phase,thereby promoting the occurrence of diapause.This study further reveals an upstream regulation mechanism for the JH signaling in insects during the reproductive diapause preparation phase,being helpful to understand the seasonal adaptation strategy of insects and providing new guidance and reference for potential target genes for pest control.