飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析

doi:10.3864/j.issn.0578-1752.2017.10.007

摘要/Abstract

摘要： 【目的】基于飞蝗（Locusta migratoria）转录组数据库获得内表皮蛋白（endocuticle structural glycoprotein）基因LmAbd-5的cDNA序列，分析该基因的序列特征和mRNA表达特性，采用RNAi方法分析其生物学功能，探讨其在飞蝗表皮形成中的作用，为害虫防治提供新的分子靶标。【方法】采用生物信息学方法搜索飞蝗转录组数据库，获得LmAbd-5 cDNA全长序列并克隆验证；采用SignalP在线软件分析蛋白的信号肽，利用SMART网站预测其功能域，使用MEGA 7.0软件中neighbor-joining（NJ）方法，与其他昆虫同源序列进行聚类分析；采用reverse-transcription quantitative PCR（RT-qPCR）方法检测LmAbd-5在飞蝗5龄第2天若虫不同组织部位和5龄不同发育时期体壁组织中的表达情况，揭示其组织和时期表达模式；采用RNA干扰（RNAi）技术及透射电镜技术（TEM）观察沉默LmAbd-5后对飞蝗生长发育和表皮结构的影响。【结果】通过搜索得到LmAbd-5 cDNA全长序列并进行了克隆和测序验证，获得520 bp全长cDNA序列，其中ORF为303 bp；基因结构分析显示该基因含有3个外显子；功能域分析发现其含有1个信号肽和1个几丁质结合域（chitin binding domain 4，ChtBD4），与沙漠蝗、白蚁、赤拟谷盗等的Abd-5结构类似；BLAST分析结果表明Abd-5在昆虫中高度保守，飞蝗与沙漠蝗Abd-5序列一致度高达81%；对其保守基序进行WebLogo分析发现LmAbd-5属于表皮蛋白CPR家族中的RR-1亚类；聚类分析结果显示，LmAbd-5与沙漠蝗和白蚁的Abd-5显示出较近的亲缘关系；RT-qPCR结果显示LmAbd-5在前肠、后肠、气管和体壁等由外胚层形成的组织中高表达，而在胃盲囊、中肠、马氏管、脂肪体和翅芽中低表达或不表达；不同时期表达分析发现，LmAbd-5在4龄若虫蜕皮后0—72 h（5龄早期）具有高表达，其中蜕皮后72 h时达到最大表达量，随后表达量急剧降低（96—168 h），其表达时期与内表皮形成时间一致；采用RNAi技术分析该基因的生物学功能，对5龄第2天若虫分别注射等量的dsLmAbd-5和dsGFP（对照），发现注射dsLmAbd-5的5龄若虫和对照组相同，均可正常蜕皮，发育至成虫第2天目的基因表达量显著降低，但未出现肉眼可见的异常表型。分别取成虫第2天处理组和对照组成虫表皮进行超微结构观察，发现与对照组相比，处理组成虫内表皮片层结构较为疏松，导致内表皮片层变厚，最终表现为整个内表皮变厚。【结论】根据转录组数据库分析获得1个CPR家族表皮蛋白LmAbd-5，该蛋白含有1个信号肽和1个几丁质结合域ChtBD4，属于RR-1亚类；LmAbd-5主要在外胚层起源的组织中高表达，沉默LmAbd-5后飞蝗没有肉眼可见的表型，但超微结构分析发现其参与飞蝗内表皮片层结构的形成。

关键词: 飞蝗, 表皮蛋白, LmAbd-5, RNAi, 透射电镜

Abstract: 【Objective】The objective of this study is to obtain a cDNA sequence of endocuticle structural glycoprotein LmAbd-5based on Locusta migratoria transcriptome, clarify its molecular characteristics and biological function, reveal its role in the formation of cuticle in L. migratoria, and provide a new molecular target for pest control. 【Method】The full length cDNAof LmAbd-5 was searched from transcriptome database of L. migratoria using bioinformatics method. The cDNA was cloned and sequenced. The signal peptide and function domain of deduced amino acid were analyzed by SignalP and SMART, respectively. Phylogenetic tree was constructed using the sequences of amino acid from different insect species by the MEGA 7.0 software with the neighbor-joining (NJ) method. Reverse transcription quantitative PCR (RT-qPCR) was applied to reveal the expression patterns of LmAbd-5 in different tissues on day 2 of 5th instar nymph and different developmental stages of integument. The effects of LmAbd-5 on locust growth development and the structure of cuticle were investigated by using RNA interference (RNAi) and transmission electron microscopy (TEM). 【Result】The full length cDNA of LmAbd-5 was got from transcriptome database, which had 520 bp including ORF 303 bp. The gene structure analysis showed that LmAbd-5 has three exons. The deduced protein contains a signal peptide and one chitin binding domain 4 (ChtBD4) through the BLAST analysis, Abd-5 was highly conserved among insect species, and the sequence identity is as high as 81% between LmAbd-5 and SgAbd-5. Abd-5 belongs to the RR-1 class of CPR family by WebLogo analysis. The result of phylogenetic tree showed that LmAbd-5 has a close genetic relationship with SgAbd-5. RT-qPCR results showed that LmAbd-5 was predominately expressed in the tissues originated from ectoderm, such as the foregut, hindgut, trachea and integument, and lower expressed or not detected in the gastric caecum, midgut, Malpighian tube, fat body and wing pads. The expression at different stages showed that LmAbd-5 mainly expressed at early of 5th instar (0-72 h after ecdysis from 4th instar nymph), and up to the peak at 72 h after molting, then markedly decreased at 96-168 h. The expression pattern is related with the formation of endocuticle. Compared with dsGFP injected control, the nymphs with the injection of dsLmAbd-5 could normally molt, and no visible abnormal phenotypes was found although the expression of LmAbd-5 was decreased significantly after dsLmAbd-5 injection. However, compared to the control group, the lamellar structure from adult cuticle with injection of dsLmAbd-5 was loose, and lamellar became thicker, finally led to the endocuticle thickening. 【Conclusion】LmAbd-5 was obtained from locust transcriptome database, which contains a signal peptide and ChtBD4, belonging to the RR-1 class of CPR family. LmAbd-5 mainly expressed in the tissues derived from ectoderm and in integument at early of 5th instar. Although there was no visible phenotypes after silencing LmAbd-5, but it was found that the lamellar structure of endocuticle is loose and endocuticle becomes thicken from ultrastructure by TEM, suggesting it may be participated in the formation of endocuticle in L. migratoria.

Key words: Locusta migratoria, cuticular proteins, LmAbd-5, RNAi, transmission electron microscopy

赵小明，贾盼，勾昕，刘卫敏，马恩波，张建珍. 飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析[J]. 中国农业科学, 2017, 50(10): 1817-1826.

ZHAO XiaoMing, JIA Pan, GOU Xin, LIU WeiMin, MA EnBo, ZHANG JianZhen. Expression and Functional Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-5 in Locusta migratoria[J]. Scientia Agricultura Sinica, 2017, 50(10): 1817-1826.

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