飞蝗海藻糖酶基因的分子特性及功能

doi:10.3864/j.issn.0578-1752.2016.22.010

摘要/Abstract

摘要： 【目的】海藻糖酶（trehalase）可专一性地将一分子海藻糖水解成为两分子葡萄糖，在昆虫能量代谢和几丁质合成过程中发挥重要作用，因此，海藻糖酶已成为害虫控制的潜在靶标。本研究以重要农业害虫飞蝗（Locusta migratoria）为材料，探讨海藻糖酶基因的分子特性及生理功能，为飞蝗的有效治理提供理论依据。【方法】通过搜索飞蝗转录组和基因组数据库，获得4个海藻糖酶基因cDNA全长序列，运用blast、TMHMM和SignalP等相关软件分析其序列特征；利用ClustalW进行海藻糖酶全长氨基酸序列比对，MEGA7构建海藻糖酶的系统进化树；运用reverse transcription-quantitative PCR（RT-qPCR）技术研究4个海藻糖酶基因在5龄若虫不同组织及发育日龄的表达特性；体外合成4个基因的dsRNA后，分别注射5龄第2天若虫，同时注射dsGFP作为对照，收集注射dsRNA 48 h后的整虫提取RNA，反转录成cDNA后，采用RT-qPCR技术检测海藻糖酶基因以及几丁质合成关键基因UDP-N-乙酰葡糖胺焦磷酸化酶基因LmUAP1和几丁质合成酶1基因LmCHS1的表达量，并观察记录表型。【结果】飞蝗4个海藻糖酶均含有海藻糖酶的2个功能保守区以及1个甘氨酸富集区，其中1个海藻糖酶具有跨膜结构域，将其命名为LmTreM（GenBank登录号：KX371563），1个海藻糖酶具有类跨膜结构，将其命名为LmTreM-like（GenBank登录号：KX371565），其余2个均为可溶性海藻糖酶，分别命名为LmTreS1和LmTreS2（GenBank登录号：KX371564和FJ795020）；系统发育分析结果显示，LmTreM和LmTreM-like与膜结合型海藻糖酶聚为一支，而LmTreS1和LmTreS2与可溶性海藻糖酶聚为一支；对4个基因在5龄若虫不同组织部位和发育日龄表达量的分析表明LmTreM、LmTreS1和LmTreS2具有组织特异表达特性，而LmTreM-like在所有组织部位中均表达，且4个海藻糖酶基因呈现出不同的发育变化趋势；RNAi结果表明，分别注射飞蝗4个海藻糖酶基因dsRNA至5龄若虫后，与对照组相比，各基因表达量均显著降低，且不存在基因间的交叉干扰，几丁质合成关键基因LmUAP1和LmCHS1的表达也无显著变化，注射各海藻糖酶基因dsRNA的5龄若虫均可成功蜕皮至成虫。【结论】飞蝗存在1个膜结合型、1个类膜结合型和2个可溶性海藻糖酶基因，这4个基因具有不同的组织和发育表达特性，任一基因的表达沉默均不影响5龄飞蝗正常蜕皮至成虫。

关键词: 飞蝗；海藻糖酶基因；表达特性；RT-qPCR, RNA干扰

Abstract: 【Objective】 Trehalase, the only enzyme that hydrolyzes one trehalose molecule into two glucose molecules, plays key roles in insect energy metabolism and chitin synthesis. So trehalase could be served as a potential target for insect pest control. In this paper, the molecular characteristics and functions of trehalase genes were explored in an important agricultural pest Locusta migratoria. The results will provide a reasonable basis for the effective management of locusts. 【Method】By searching the transcriptome and genome of L. migratoria, four full-length cDNA of trehalase genes were obtained. Sequence characteristics of these four trehalases were analyzed by using blast, TMHMM and SignalP softwares. Multiple amino acid sequence alignment of trehalases was made using ClustalW. The phylogenetic tree was constructed by MEGA 7. The expression patterns of trehalase genes in different tissues and developmental days were studied in the 5th-instar nymphs by RT-qPCR. The dsRNAs of four trehalase genes were synthesized in vitro, and then injected into the 5th-instar nymphs on day 2, respectively. Control nymphs were injected with dsGFP alone. The whole body after the dsRNA injection for 48 h was used for total RNA extraction and cDNA synthesis. RT-qPCR were performed to determine the transcript levels of four trehalase genesand genes involved in chitin synthesis such as UDP-N-acetylglucosamine pyrophosphorylase gene LmUAP1 andchitin synthase 1 gene LmCHS1. The abnormal nymphs displayed phenotypes were carefully observed. 【Result】 Blast analysis showed that all these four trehalases contained two conservative regions and a glycine enrichment region. A membrane-bound trehalase with a typical transmembrane domain was named LmTreM (GenBank accession number KX371563). A transmembrane like domain was predicted in another trehalase, named LmTreM-like (GenBank accession number KX371565). The two remains were soluble trehalases, named LmTreS1 and LmTreS2, respectively (GenBank accession number KX371564 and FJ795020). Phylogenetic analysis showed that LmTreM and LmTreM-like, LmTreS1 and LmTreS2 were clustered with membrane-bound and soluble trehalases, respectively. RT-qPCR was carried out to analyze the expression patterns of four trehalase genes in different tissues and developmental days in the 5th-instar nymphs of L. migratoria. The results indicated LmTreM, LmTreS1 and LmTreS2 were mainly expressed in specific tissues, and LmTreM-like was consistently expressed in all selected tissues. mRNA transcripts of four trehalase genes were different during the development of 5th-instar nymphs. RNAi results suggested that expressions of four trehalase genes were significantly reduced compared to the control, and the injection of LmTreM, LmTreS1, LmTreS2 and LmTreM-like dsRNA only repressed the expression of the targeted LmTre without reducing that of other LmTres, the expressions of LmUAP1 and LmCHS1 involved in chitin synthesis were not affected. However, nymphs could successfully molt to adults after injected with four trehalase dsRNAs, respectively.【Conclusion】One membrane-bound, one membrane-bound like and two soluble trehalase were found in L. migratoria. The four trehalase genes showed different tissue and developmental expression characteristics, silencing of each of these four trehalase genes in the 5th-instar nymphs could not affect the normal molting to adults.

Key words: Locusta migratoria, trehalase gene, expression characteristics, RT-qPCR, RNA interference

刘晓健，孙亚文，崔淼，马恩波，张建珍. 飞蝗海藻糖酶基因的分子特性及功能[J]. 中国农业科学, 2016, 49(22): 4375-4386.

LIU Xiao-jian, SUN Ya-wen, CUI Miao, MA En-bo, ZHANG Jian-zhen. Molecular Characteristics and Functional analysis of Trehalase Genes in Locusta migratoria

[J]. Scientia Agricultura Sinica, 2016, 49(22): 4375-4386.

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