中华稻蝗羧酸酯酶家族基因生物信息学及组织表达特异性分析

doi:10.3864/j.issn.0578-1752.2015.21.008

摘要/Abstract

摘要： 【目的】对重要农业害虫中华稻蝗（Oxya chinensis）羧酸酯酶（carboxylesterases，Ces）家族基因进行生物信息学分析，并对部分基因的组织表达特性进行研究，为揭示中华稻蝗Ces基因功能及研发新的分子靶标提供依据。【方法】基于中华稻蝗转录组数据库，通过关键词搜索获得Ces基因序列，采用生物信息学方法进行比对拼接、氨基酸序列推导和开放阅读框分析，选择全长序列构建系统发育树，采用在线软件分析中华稻蝗Ces氨基酸序列结构、理化性质、信号肽和N糖基化位点等特征。采用实时定量PCR技术，通过geNorm 与Normfinder 软件分析4个候选内参基因β-肌动蛋白（β-actin）、延长因子（EF1α）、3-磷酸甘油醛脱氢酶（GAPDH）和核糖体蛋白49（RP49）表达稳定性，筛选中华稻蝗5龄若虫不同组织部位最适内参基因并检测10个羧酸酯酶基因在不同组织部位相对表达量。【结果】从中华稻蝗转录组数据库搜索获得Ces基因cDNA片段180个，经筛选获得28个Ces基因全长序列进行生物信息学分析。推导的氨基酸序列与其他昆虫物种的聚类分析结果显示，中华稻蝗28个Ces分布于4个功能簇中，其中A簇直翅目和部分双翅目昆虫α酯酶（20个）、D簇表皮特异酯酶（2个）、E簇β酯酶（4个）和F簇非鳞翅目保幼激素酯酶（2个）。氨基酸序列分析结果显示，中华稻蝗大部分Ces的等电点（pI）均为偏酸性或弱酸性，pI为4.38—6.84，只有3个Ces的pI值偏碱性。中华稻蝗所有的Ces基因都具有N糖基化位点、N端保守的半胱氨酸残基及氧离子洞，信号肽预测结果表明19个Ces具有完整的信号肽；21个Ces具有典型的催化三联体S-E-H，其余7个Ces催化三联体发生了不同的替换。GeNorm与Normfinder分析结果显示，4个候选内参基因稳定性EF1α=RP49＞β-actin＞GAPDH。选择EF1α作为内参基因，RT-qPCR结果显示10个羧酸酯酶基因在7个不同组织部位均有表达，其中OcCesA1、OcCesA6、OcCesA12、OcCesA14、OcCesA18和OcCesA19在中肠和胃盲囊高表达，此外OcCesA6、OcCesA18和OcCesA19还在马氏管高表达，OcCesA2在后肠高表达。OcCesD1在马氏管表达量最高，脂肪体和表皮次之；OcCesE1在胃盲囊表达量最高；OcCesF2在中肠表达量最高。【结论】基于中华稻蝗转录组数据库获得28个全长Ces基因，聚类分析结果显示，这些基因分布于4个功能簇中，其中分布于A簇的基因数目相对较多，且多在中肠、胃盲囊和马氏管等代谢解毒器官中高表达。研究结果为Ces基因功能的深入探索和中华稻蝗Ces基因分子靶标研发提供了依据。

关键词: 中华稻蝗, 转录组, 羧酸酯酶基因, 生物信息学, 组织特异表达

Abstract: 【Objective】Oxya chinensis is an important agricultural pest. The objective of this study is to reveal the function of carboxylesterases (Ces) genes and select potential molecular target for pest management through conducting the bioinformatics and tissue-specific expression analysis of Ces genes from O. chinensis transcriptome. 【Method】 The cDNA sequences of Ces genes were searched using the keyword “carboxylesterase” against the O. chinensis transcriptome database. The Ces cDNA sequence assembling, the deduced amino acid sequence analysis and the open reading frame finding were performed by using bioinformatics methods. Phylogenetic tree of O. chinensis carboxylesterase with complete ORF was constructed using neighbor-joining method. The structure, calculated molecular mass, isoelectric points, signal peptide and potential N-glycosylation sites of O. chinensis Ces amino acid sequences were predicted using corresponding online softwares. Optimal reference genes in different tissues of 5th instar nymphs were screened and relative expressions in different tissues of 10 Ces genes were detected by reverse transcription- quantitative PCR (RT-qPCR).【Result】 A total of 180 putative Ces cDNA fragments were identified from O. chinensis transcriptome. Among them 28 full-length cDNAs containing complete ORFs were obtained. Phylogenetic tree of Ces protein sequences from O. chinensis and other insect species showed that 28 O. chinensis Ces protein sequences were distributed in four clades, including clade A with Orthopteran and part of Dipteran α-esterases (20 Ces protein sequences), clade D with integumental esterases (2), clade E with β-esterases (4), and clade F with nonlepidopteran JHEs (2). With the exception of three Ces proteins with alkaline isoelectric points (pI), all the remaining Ces proteins are acidic or slightly acidic with pI ranging from 4.38 to 6.84. All the Ces protein sequences have N-glycosylation site, N-terminal conserved cysteine residues, and oxyanion hole. Signal peptide prediction showed 19 of 28 have signal peptide. Twenty-one Ces protein sequences have conserved catalytic triad S-E-H, however, the remaining 7 Ces protein sequences showed different amino acid substitutions. GeNorm and Normfinder analysis showed that EF1α and RP49 were the optimal reference genes. RT-qPCR showed that 10 Ces genes were expressed in 7 different tissues, in which OcCesA1, OcCesA6, OcCesA12, OcCesA14, OcCesA18 and OcCesA19 mainly expressed in the midgut and gastric caecum; OcCesA6, OcCesA18, OcCesA19 still expressed in Malpighian tubules; OcCesA2 highly expressed in the hindgut. OcCesD1 had the highest expression in Malpighian tubules, followed by the fat body and integument. OcCesE1 had the highest expression in gastric caeca, OcCesF2 had the highest expression in midgut.【Conclusion】A total of 28 Ces cDNAs with complete ORFs were identified from the O. chinensis transcriptome database. These Ces cDNAs were clustered into four different clades in the phylogenetic analysis. Significant expansions of the Ces genes were found in clade A and highly expressed in detoxification, excretory organs, such as midgut, gastric caeca and Malpighian tubules. These results contributed to the further functional analysis of O. chinensis Ces and biological control of O. chinensis based on Ces gene molecular target.

Key words: Oxya chinensis, transcriptome, carboxylesterase gene, bioinformatics, tissue-specific expression

刘娇，张建珍，李大琪，张婷婷，马恩波，张建琴. 中华稻蝗羧酸酯酶家族基因生物信息学及组织表达特异性分析[J]. 中国农业科学, 2015, 48(21): 4272-4284.

LIU Jiao, ZHANG Jian-zhen, LI Da-qi, ZHANG Ting-ting, MA En-bo, ZHANG Jian-qin. Bioinformatics and Tissue-Specific Expression Analysis of Carboxylesterase Genes from Oxya chinensis[J]. Scientia Agricultura Sinica, 2015, 48(21): 4272-4284.

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