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LmTre(GenBank M登录号:KX371563),1个海藻糖酶具有类跨膜结构,将其命名为Lm Tre M-like(Gen Bank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�
【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 Signal P softwares. Multiple amino acid sequence alignment of trehalases was made using Clustal W. 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 ds RNA injection for 48 h was used for total RNA extraction and cDNA synthesis. RT-q PCR were performed to determine the transcript levels of four trehalase genes and genes involved in chitin synthesis such as UDP-N-acetylglucosamine pyrophosphorylase gene Lm UAP1 and chitin 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 Lm Tre M（Gen Bank accession number KX371563）. A transmembrane like domain was predicted in another trehalase, named Lm Tre M-like（Gen Bank accession number KX371565）. The two remains were soluble trehalases, named LmTreS1 and LmTreS2, respectively（Gen Bank accession number KX
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第22期4375-4386,共12页
Scientia Agricultura Sinica
基金
国家自然科学基金(31402020
31272380)
山西省高校科技创新(2016113)
山西省青年基金(201601D021120)
山西省科技基础条件平台建设(2015091010)
