飞蝗表皮蛋白Obstructor家族基因的分子特性及基于RNAi的功能分析  

Molecular Characterization and RNAi-Based Functional Analysis of Obstructor Family Genes in Locusta migratoria

作　　者：王燕[1,2] 李大琪[1,2] 刘晓健[1,2] 李涛[1,2] 马恩波[1,2] 范仁俊[2,3] 张建珍[1,2] 

机构地区：[1]山西大学应用生物学研究所,太原030006 [2]农业有害生物综合治理山西省重点实验室,太原030006 [3]山西省农业科学院植物保护研究所,太原030031

出　　处：《中国农业科学》2015年第1期73-82,共10页Scientia Agricultura Sinica

基　　金：国家"973"计划(2012CB114102);国家自然科学基金(31272380);教育部博士点专项基金(博导类)(20121401110008)

摘　　要：【目的】获得飞蝗(Locusta migratoria)表皮蛋白Obstructor(Obst)家族基因的c DNA序列,并研究其序列特征和m RNA表达特性,探讨其生物学功能,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Obst家族基因c DNA片段,并进行BLAST分析得到Obst家族基因的c DNA序列;采用RACE技术扩增该家族基因的3′c DNA序列,拼接后获得全长;Signal P在线软件分析蛋白的信号肽,SMART网站预测其功能域,并使用Mega 5.10软件中Neighbor-Joining方法,与黑腹果蝇(Drosophila melanogaster)Obst家族基因和赤拟谷盗(Tribolium castaneum)CPAP3家族基因(Obst家族基因的同源基因)氨基酸序列进行聚类分析;采用real-time quantitative PCR(q PCR)方法检测Lm Obst家族基因在5龄若虫不同组织部位和不同龄期体壁的表达情况,绘制表达图谱;采用RNA干扰(RNAi)技术探讨Lm Obsts对飞蝗发育的影响。【结果】在飞蝗转录组数据库中搜索得到8个Obst家族基因的c DNA片段,通过NCBI进行BLAST分析显示与赤拟谷盗CPAP3、黑腹果蝇Obst高度同源,属于Lm Obst家族基因片段,其中5个是全长序列,3个序列缺失3′端;采用RACE技术获得3′末端c DNA序列;将得到的8个Lm Obst家族基因全长序列进行功能域分析,发现具有Obst家族表皮蛋白的特点,即有1个信号肽与3个几丁质结合域Cht BD2;并与黑腹果蝇、赤拟谷盗同源基因进行进化树的构建,根据进化树分析结果,分别命名为Lm Obst-A1、Lm Obst-A2、Lm Obst-B、Lm Obst-C、Lm Obst-D1、Lm Obst-D2、Lm Obst-E1和Lm Obst-E2。q PCR结果显示Lm Obst-E1和Lm Obst-E2在前肠和后肠高特异性表达,Lm Obst-D1在体壁和前肠高表达,其他Lm Obsts在体壁或外胚层内陷形成的前肠和后肠高表达,在胃盲囊、中肠、马氏管和脂肪体中低表达;Lm Obst家族基因在5龄若虫不同天数体壁的表达趋势比较接近,在5龄前期高表达,中期降到最低,蜕皮前又上升到高�Objective] The objective of this study is to obtain cDNA sequences of Obstructor （Obst） family genes of cuticular proteins from Locusta migratoria, clarify their molecular characterization and biological function, and to get new molecular target for pest management. [Method] The cDNA fragments of Obst family genes were searched from locust transcriptome database and the sequences were further analyzed by BLAST at NCBI, the candidated cDNA fragments belonging to the Obst family genes were confirmed by sequence alignment with other known insect Obst family genes. The primers were designed for non full-length cDNA sequences and RACE-PCR was performed to amplify 3′ cDNA sequences, the full-length cDNA sequences of Obst family genes were assembled by overlap region. All the full-length cDNA sequences were translated into amino acid sequences and signal peptides were analyzed by SignalP tool, functional domains were predicted by SMART website. Then phylogenetic tree was constructed by using Mega 5.10 software with homologous amino acid sequences encoded by the Obsts from Drosophila melanogaster and the CPAP3 family genes （the Obst homologous genes） from Tribolium castaneum. The real-time quantitative PCR （qPCR） was applied to analyze the gene expression patterns of LmObst genes in different tissues and developmental stages of the 5th instar nymphs in the integument. RNA interference （RNAi） technology was used to explore biological function of these Obst genes. [Result] Eight cDNA fragments assumed to be Obst family genes were got from locust transcriptome database, which showed high similarity with CPAP3 family genes of T. castaneum and Obst family genes of D. melanogaster by BLAST analysis. Five of them were the full-length cDNA sequences, and three of them missed 3′ end sequences. Then three full-length cDNA sequences were further amplified by using RACE-PCR technique. The functional domain analysis of eight LmObsts showed that they all had a signal peptide and three chitin binding domain ChtBD2,

关 键 词：飞蝗 表皮蛋白 Obstructor real-time QUANTITATIVE PCR RNAI 

分 类 号：S433.2[农业科学—农业昆虫与害虫防治]