荔枝蒂蛀虫信息素结合蛋白基因序列及表达分析

doi:10.16380/j.kcxb.2018.05.002

摘要/Abstract

摘要： 【目的】获得荔枝蒂蛀虫Conopomorpha sinensis 3个信息素结合蛋白（PBP）基因的全长序列，并分析序列和表达特征，为更好地利用性信息素防治该虫提供必要的基础。【方法】提取荔枝蒂蛀虫触角总RNA，利用转录组测序结果和RACE-PCR技术获得3个PBP基因的全长序列；对序列进行生物信息学分析，用I-TASSER在线软件建立蛋白三维模型，用TM-align软件进行同源建模，用COACH软件推测蛋白的结合位点；用荧光定量PCR方法分析这3个基因在不同龄期（幼虫和蛹）和3日龄雌雄成虫不同组织［触角、头（去除触角）、胸、腹、足和翅］中的表达谱。【结果】从荔枝蒂蛀虫触角中克隆了3个PBP基因的全长序列，分别命名为CsinPBP1, CsinPBP2和CsinPBP3（GenBank登录号： MF093145, MF093146和MF093147）。序列分析结果表明，这3个基因的编码蛋白具有昆虫气味结合蛋白的典型特征，并且CsinPBP1与紫色卷蛾Yponomeuta cagnagellus PBP的氨基酸序列一致性达72%，CsinPBP2与小菜蛾Plutella xylostella PBP1的氨基酸序列一致性达55%，CsinPBP3与水稻大螟Sesamia inferens PBP3的氨基酸序列一致性达39%。软件模拟分析表明，CsinPBP1, CsinPBP2和CsinPBP3蛋白的三维结构分别与家蚕Bombyx mori普通气味结合蛋白2（GOBP2）（PDB: 2wc6A）、脐橙螟Amyetois transitetella PBP1（PDB: 4inxA）和家蚕PBP（PDB: 2p70A）相似度最高，分别预测得到10, 7和8个结合位点。表达谱分析显示，3个基因均只在成虫触角中表达，在幼虫期和蛹期不表达，在成虫头（去除触角）、胸、腹、足和翅中也不表达，且在雄虫触角中的表达量分别是雌虫中表达量的1.94, 28.19和32.94倍。【结论】获得荔枝蒂蛀虫3个PBP基因的全长序列，序列和表达分析结果提示这3个基因与雄虫感受性信息素有关。

关键词: 荔枝蒂蛀虫, 信息素结合蛋白, 基因克隆, 序列分析, 软件模拟, 表达谱

Abstract: 【Aim】 This study aims to clone three pheromone binding protein (PBP) genes from the litchi fruit borer, Conopomorpha sinensis, and to analyze their sequences and expression characteristics, so as to provide essential basis for better use of sex pheromone for control. 【Methods】 The full-length cDNA of three PBP genes were cloned from the antennae of C. sinensis adults by transcriptome and RACE-PCR technique after extracting total RNA. The putative amino acid sequences were analyzed by bioinformatics software. The software I-TASSER was used to simulate 3D models, the software TM-align was used in protein homology modeling and the software COACH was used to speculate the binding sites. The expression profiles of the three genes in different developmental stages (larval and pupal stages), and different tissues of the 3 d-old female and male adults (antennae, head without antennae, thorax, abdomen, legs and wings) were analyzed by real-time PCR. 【Results】 Three PBP genes were cloned from the antennae of C. sinensis adults. They were named CsinPBP1, CsinPBP2 and CsinPBP3, and deposited under GenBank accession numbers MF093145, MF093146 and MF093147, respectively. Bioinformatic analysis revealed that the encoded proteins of the three genes have typical characteristics of odor binding proteins. Phylogenetic analysis revealed that CsinPBP1 has 72% amino acid sequence identity with PBP of Yponomeuta cagnagellus, CsinPBP2 has 55% amino acid sequence identity with PBP1 of Plutella xylostella, and CsinPBP3 has 39% amino acid sequence identity with PBP3 of Sesamia inferens. Software simulation analysis revealed that the 3D structures of CsinPBP1, CsinPBP2 and CsinPBP3 were the most similar with GOBP2 of Bombyx mori (PDB: 2wc6A), PBP1 of Amyetois transitetella (PDB: 4inxA) and PBP of B. mori (PDB: 2p70A), and were predicted to have 10, 7 and 8 binding sites, respectively. Expression profiling revealed that the three genes were only expressed in adult antennae, but neither in larval and pupal stages nor in other adult tissues including the head without antennae, thorax, abdomen, legs and wings. The relative expression levels of CsinPBP1, CsinPBP2 and CsinPBP3 in the antennae of male adults were 1.94, 28.19 and 32.94 times as high as those in the antennae of male adults, respectively. 【Conclusion】 Three PBP genes were cloned in C. sinensis. Their sequence and expression analysis suggest that they are related to sex pheromone sensing in males.

Key words: Conopomorpha sinensis, pheromone binding protein, gene cloning, sequence analysis, software simulation, expression profile

李鹏燕, 刘艳萍, 王思威, 孙海滨, 柏建山, 彭刚, 龚雪海. 荔枝蒂蛀虫信息素结合蛋白基因序列及表达分析[J]. , 2018, 61(5): 527-536.

LI Peng-Yan, LIU Yan-Ping, WANG Si-Wei, SUN Hai-Bin, BAI Jian-Shan, PENG Gang, GONG Xue-Hai. Sequence analysis and expression profiling of pheromone binding protein genes in the litchi fruit borer, Conopomorpha sinensi (Lepidoptera: Gracillariidae)[J]. , 2018, 61(5): 527-536.

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链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2018.05.002

http://www.insect.org.cn/CN/Y2018/V61/I5/527

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荔枝蒂蛀虫信息素结合蛋白基因序列及表达分析

Sequence analysis and expression profiling of pheromone binding protein genes in the litchi fruit borer, Conopomorpha sinensi (Lepidoptera: Gracillariidae)

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