草履蚧触角嗅觉基因转录组及生物信息学分析
发布时间：2018-09-01 18:59
【摘要】：草履蚧Drosicha corpulenta(Kuwana)(半翅目:蚧总科:绵蚧科),是一种聚集在枝芽处刺吸汁液取食的植食性昆虫,当虫口密度过大时往往会造成枝芽枯死。由于草履蚧体表分泌蜡质形成保护层,人工清除和化学防治难以对其进行有效控制,而大量化学杀虫剂的使用不仅会灭杀天敌,还会对果实和环境造成污染。因此本文从昆虫化学通讯对害虫防治的角度出发,对草履蚧触角中参与嗅觉形成过程的相关蛋白进行研究,发现嗅觉基因在蚧虫中的作用,为今后的生物防治提供新的视角及理论依据。本文的研究内容包括:(1)对草履蚧雌成虫触角转录组进行测序,并进行组装、注释和同源性分析;从Unigenes中预测草履蚧嗅觉基因并归纳基因信息;对草履蚧的气味结合蛋白(OBPs)及化学感受蛋白(CSPs)序列进行比对,并构建系统发育树;(2)在转录组测序和分析的基础上,得到气味结合蛋白OBP(DcorOBP4,5,6,7,8,14)和化学感受蛋白DcorCSP2的编码序列;利用生物信息学方法分析其序列信息,并构建三维结构。结果如下:1.本文对草履蚧雌成虫触角转录组进行高通量测序。在转录组中,共获得33,741,845条序列标签reads,其中16,334条非重复序列unigenes被组装。这些unigenes通过GO分析和eggNOG分析进行基因功能的分类统计。草履蚧触角中unigenes与其他物种相似性比较发现,与豌豆蚜相似的数量最多,达到18.31%。2.本文从unigenes中预测到16条编码气味结合蛋白OBPs的基因、2条编码化学感受蛋白CSPs的基因、1条编码感觉神经元膜蛋白SNMP的基因、21条气味受体基因ORs和7条编码味觉受体GRs的基因,并总结归纳了 47条嗅觉基因信息。此外,对草履蚧触角中预测的气味结合蛋白OBPs和化学感受蛋白CSPs序列与蚜虫、盲蝽和扶桑绵粉蚧相关序列构建系统发育树。结果显示,8条气味结合蛋白与蚜虫聚在一起,3条草履蚧气味结合蛋白OBPs聚在一起。2条化学感受蛋白与扶桑绵粉蚧的2条化学感受蛋白分别聚类在一起。根据聚在一起且已知其功能的蚜虫和盲蝽的物种,可以对草履蚧的OBPs功能进行预测。草履蚧嗅觉基因的发现对认识其在蚧虫行为反应中的作用提供新的视角,同时提高蚧虫的生物防治策略。3.本研究在草履蚧雌成虫触角转录组测序和分析的基础上,通过RT-PCR技术得到的气味结合蛋白OBPs(DcorOBP4,5,6,7,8,14)和化学感受蛋白DcorCSP2的编码序列,利用生物信息学的方法对草履蚧这些蛋白的理化性质、信号肽、亲疏水性等进行预测分析。分析表明,这些蛋白均有完整的开放阅读框ORF,N端带有19～29个氨基酸的信号肽,等电点在4.12～9.30之间,相对分子质量为15.6～17.0KDa,这些蛋白均为亲水性蛋白。OBPs(DcorOBP4,5,6,7,8,14)含有六个保守的半胱氨酸位点,符合典型的气味结合蛋白家族的特征。化学感受蛋白DcorCSP2包含有四个保守的半胱氨酸位点,符合化学感受蛋白家族的特征。草履蚧典型的气味结合蛋白(DcorOBP4,5,6,7,8,14)和化学感受蛋白DcorCSP2通过同源建模后发现,三维结构都是由六个α螺旋组成。DcorOBP4,6,7,8这四个气味结合蛋白的六个保守半胱氨酸形成了三对二硫键,与典型的气味结合蛋白特征相符。化学感受蛋白DcorCSP2也构建出两对二硫键,这些二硫键均起到稳定蛋白质结构的作用。以上6条OBPs均由α1,α2,α4,α5,α6这5个α螺旋形成疏水口袋,α3在口袋的一端不参与疏水口袋的形成。此外,这些蛋白的C-末端均折合进入蛋白质的口袋内部,使得口袋结构变得更加稳固。
[Abstract]:Drosicha corpulenta (Kuwana) is a kind of herbivorous insect that gathers in the shoots and sucks juice. When the population density is too high, it often causes the shoots to wither. Quantitative chemical insecticides not only kill natural enemies, but also cause pollution to fruits and environment. Therefore, from the point of view of insect chemical communication for pest control, this paper studied the proteins involved in olfactory formation in the antennae of paramecium, and found the role of olfactory genes in scale insects, providing a new way for biological control in the future. The main contents of this study include: (1) Sequencing, assembly, annotation and homology analysis of antennal transcriptome of female Paramecium paramecium; Predicting olfactory genes of Paramecium from Unigenes and inducing gene information; aligning and constructing odorant binding protein (OBPs) and chemosensory protein (CSPs) sequences of Paramecium paramecium. Establishing phylogenetic trees; (2) On the basis of transcriptome sequencing and analysis, the coding sequences of odorant binding protein OBP (DcorOBP 4, 5, 6, 7, 8, 14) and chemosensory protein DcorCSP 2 were obtained; the sequence information was analyzed by bioinformatics method, and the three-dimensional structure was constructed. The results were as follows: 1. Sequencing. A total of 33,741,845 reads were obtained in the transcriptome, of which 16,334 non-repetitive unigenes were assembled. These unigenes were classified and counted by GO analysis and eggNOG analysis. The similarity of unigenes in the antennae of Paramecium paramecis to other species was the highest, reaching 18.31%. 16 genes encoding odorant binding protein OBPs, 2 genes encoding chemoreceptor protein CSPs, 1 gene encoding sensory neuron membrane protein SNMP, 21 odorant receptor genes ORs and 7 genes encoding taste receptor GRs were predicted from unigenes, and 47 olfactory genes were summarized. The predicted odor-binding proteins OBPs and chemosensory proteins CSPs were sequenced to construct phylogenetic trees associated with aphids, bugs and Fusang mealworms. The results showed that eight odor-binding proteins were clustered with aphids, and three odor-binding proteins OBPs were clustered with paramecium. Two chemosensory proteins and two chemosensory eggs of Fusang mealworm were clustered with two chemosensory proteins. The OBPs function of Paramecium can be predicted according to the species of aphids and bugs that are clustered together and whose functions are known. The discovery of the olfactory genes of Paramecium provides a new perspective for understanding their role in behavioral responses of scale insects and improves the biological control strategies of scale insects. 3. This study was conducted in female Paramecium caterpillars. On the basis of antennal transcriptome sequencing and analysis, the coding sequences of odor-binding proteins OBPs (DcorOBP4, 5, 6, 7, 8, 14) and chemosensory proteins DcorCSP2 were obtained by RT-PCR. The physicochemical properties, signal peptides, hydrophilicity and hydrophobicity of these proteins were predicted by bioinformatics method. All of them have complete open reading frame ORF, N-terminal with 19-29 amino acid signal peptide, isoelectric point between 4.12-9.30, relative molecular weight of 15.6-17.0 KDa, these proteins are hydrophilic proteins. OBPs (DcorOBP4, 5, 6, 7, 8, 14) contain six conserved cysteine sites, which conform to the characteristics of typical odor binding protein family. The receptor protein DcorCSP2 contains four conserved cysteine sites, which conform to the characteristics of the chemoreceptive protein family. Typical odorant-binding proteins (DcorOBP4, 5, 6, 7, 8, 14) and chemoreceptive proteins (DcorCSP2) of Paramecium indicum were homologously modeled and found to be composed of six alpha-helices. The six conserved cysteines formed three pairs of disulfide bonds, which were consistent with the characteristics of typical odor-binding proteins. The chemosensory protein DcorCSP2 also constructed two pairs of disulfide bonds. These disulfide bonds stabilized the structure of proteins. All the six OBPs formed hydrophobic pockets from the five alpha helixes of alpha 1, alpha 2, alpha 4, alpha 5, and alpha 6. Alpha 3 was located at one end of the pocket. It is not involved in the formation of hydrophobic pockets. In addition, the C-terminals of these proteins are folded into the inner pockets of the proteins, making the structure of the pockets more stable.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433;Q78
本文编号：2218065
[Abstract]:Drosicha corpulenta (Kuwana) is a kind of herbivorous insect that gathers in the shoots and sucks juice. When the population density is too high, it often causes the shoots to wither. Quantitative chemical insecticides not only kill natural enemies, but also cause pollution to fruits and environment. Therefore, from the point of view of insect chemical communication for pest control, this paper studied the proteins involved in olfactory formation in the antennae of paramecium, and found the role of olfactory genes in scale insects, providing a new way for biological control in the future. The main contents of this study include: (1) Sequencing, assembly, annotation and homology analysis of antennal transcriptome of female Paramecium paramecium; Predicting olfactory genes of Paramecium from Unigenes and inducing gene information; aligning and constructing odorant binding protein (OBPs) and chemosensory protein (CSPs) sequences of Paramecium paramecium. Establishing phylogenetic trees; (2) On the basis of transcriptome sequencing and analysis, the coding sequences of odorant binding protein OBP (DcorOBP 4, 5, 6, 7, 8, 14) and chemosensory protein DcorCSP 2 were obtained; the sequence information was analyzed by bioinformatics method, and the three-dimensional structure was constructed. The results were as follows: 1. Sequencing. A total of 33,741,845 reads were obtained in the transcriptome, of which 16,334 non-repetitive unigenes were assembled. These unigenes were classified and counted by GO analysis and eggNOG analysis. The similarity of unigenes in the antennae of Paramecium paramecis to other species was the highest, reaching 18.31%. 16 genes encoding odorant binding protein OBPs, 2 genes encoding chemoreceptor protein CSPs, 1 gene encoding sensory neuron membrane protein SNMP, 21 odorant receptor genes ORs and 7 genes encoding taste receptor GRs were predicted from unigenes, and 47 olfactory genes were summarized. The predicted odor-binding proteins OBPs and chemosensory proteins CSPs were sequenced to construct phylogenetic trees associated with aphids, bugs and Fusang mealworms. The results showed that eight odor-binding proteins were clustered with aphids, and three odor-binding proteins OBPs were clustered with paramecium. Two chemosensory proteins and two chemosensory eggs of Fusang mealworm were clustered with two chemosensory proteins. The OBPs function of Paramecium can be predicted according to the species of aphids and bugs that are clustered together and whose functions are known. The discovery of the olfactory genes of Paramecium provides a new perspective for understanding their role in behavioral responses of scale insects and improves the biological control strategies of scale insects. 3. This study was conducted in female Paramecium caterpillars. On the basis of antennal transcriptome sequencing and analysis, the coding sequences of odor-binding proteins OBPs (DcorOBP4, 5, 6, 7, 8, 14) and chemosensory proteins DcorCSP2 were obtained by RT-PCR. The physicochemical properties, signal peptides, hydrophilicity and hydrophobicity of these proteins were predicted by bioinformatics method. All of them have complete open reading frame ORF, N-terminal with 19-29 amino acid signal peptide, isoelectric point between 4.12-9.30, relative molecular weight of 15.6-17.0 KDa, these proteins are hydrophilic proteins. OBPs (DcorOBP4, 5, 6, 7, 8, 14) contain six conserved cysteine sites, which conform to the characteristics of typical odor binding protein family. The receptor protein DcorCSP2 contains four conserved cysteine sites, which conform to the characteristics of the chemoreceptive protein family. Typical odorant-binding proteins (DcorOBP4, 5, 6, 7, 8, 14) and chemoreceptive proteins (DcorCSP2) of Paramecium indicum were homologously modeled and found to be composed of six alpha-helices. The six conserved cysteines formed three pairs of disulfide bonds, which were consistent with the characteristics of typical odor-binding proteins. The chemosensory protein DcorCSP2 also constructed two pairs of disulfide bonds. These disulfide bonds stabilized the structure of proteins. All the six OBPs formed hydrophobic pockets from the five alpha helixes of alpha 1, alpha 2, alpha 4, alpha 5, and alpha 6. Alpha 3 was located at one end of the pocket. It is not involved in the formation of hydrophobic pockets. In addition, the C-terminals of these proteins are folded into the inner pockets of the proteins, making the structure of the pockets more stable.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433;Q78
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