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

摘要

【目的】获得飞蝗（Locusta migratoria）表皮蛋白 Obstructor（Obst）家族基因的 cDNA 序列，并研究其序列特征和 mRNA 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 cDNA 片段，并进行 BLAST 分析得到 Obst 家族基因的 cDNA 序列；采用 RACE 技术扩增该家族基因的3′cDNA 序列，拼接后获得全长；SignalP 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（qPCR）方法检测 LmObst 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 LmObsts 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cDNA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 LmObst 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 cDNA 序列；将得到的8个 LmObst 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 ChtBD2；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 LmObst-A1、LmObst-A2、LmObst-B、LmObst-C、LmObst-D1、LmObst-D2、LmObst-E1和 LmObst-E2。qPCR 结果显示 LmObst-E1和 LmObst-E2在前肠和后肠高特异性表达，LmObst-D1在体壁和前肠高表达，其他 LmObsts 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；LmObst 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，蜕皮前又上升到高水平。采用 RNAi 技术研究基因的生物学功能，5龄若虫第5天分别注射 dsLmObst，对照组注射等量 dsGFP，48 h 后检测沉默效率，发现目的基因表达量显著降低；进一步观察发现，注射 dsLmObst-E1的5龄若虫80%无蜕皮迹象，在5龄若虫状态下死亡，剩余20%若虫蜕皮延迟1—2 d，并且蜕至成虫后，16 h 内全部死亡；其余注射双链的试虫普遍发生蜕皮延迟1—3 d 的现象，但未发现其他可见的异常表型。【结论】获得8个飞蝗 LmObst家族基因，所有 Obst 家族蛋白功能域保守，均有1个信号肽与3个几丁质结合域 ChtBD2；LmObsts 主要参与飞蝗体壁和前、后肠等外胚层发育来源组织器官的形成。LmObst-E1是飞蝗发育所必需的，该基因沉默可导致飞蝗死亡，其他7个 LmObsts 的沉默导致飞蝗发育延迟1-3 d，但无致死效应。%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, which are in accordance with the characteristics of the representative insect Obst cuticular protein. According to the result of phylogenetic analysis, eight LmObst genes were named as LmObst-A1, LmObst-A2, LmObst-B, LmObst-C, LmObst-D1, LmObst-D2, LmObst-E1 and LmObst-E2, respectively. The qPCR results showed the tissue distribution of eight LmObst genes. LmObst-E1 and LmObst-E2 were specifically expressed in foregut and hindgut, LmObst-D1 was mainly expressed in integument and foregut, the others were highly expressed in integument, foregut and hindgut, and lowly expressed in gastric caeca, midgut, Malpighian tube and fat body. Developmental expression patterns showed that they had a similar trend, eight LmObst genes were highly expressed at the early stage of the 5th instar nymphs, gradually reduced to a minimum at the middle stage, then got raised before molting. RNAi was applied to explore their biological function, eight dsLmObsts were injected into the 5th day of 5th instar nymphs, respectively, and the control group was injected with equal amount dsGFP. The silencing efficiency was detected at 48 h after dsRNA injection, and the mRNA expression was significantly reduced. Further phenotypic observation showed that 80% nymphs injected with dsLmObst-E1 appeared no molting and died, the rest 20% nymphs could molt to the next stage, but the molting time delayed about 1-2 d, after molting to adults, the development was retarded and died within 16 h. However, the control nymphs with dsGFP injection successfully molted to the adults and developed well. The nymphs injected with the other dsObsts displayed slow development, the molting time delayed about 1-3 d, but no visible abnormal phenotypes were found. [Conclusion] The eight full-length cDNA sequences of Obst family genes were obtained from L. migratoria. LmObsts had typical domain structure of Obst protein with a signal peptide and three chitin binding domain (ChtBD2). LmObsts were mainly responsible for the formation of integument, foregut and hindgut, which developed from ectoderm. LmObst-E1 is essential for locust development, silencing LmObst-E1 was lethal to L. migratoria, the mRNA suppression of the other seven LmObsts displayed developmental delay, but no lethal effect was observed.