绿盲蝽气味结合蛋白OBPs基因的鉴定及AlucOBP13、AlucOBP17的气味结合特性

发布时间：2018-01-16 23:35

本文关键词：绿盲蝽气味结合蛋白OBPs基因的鉴定及AlucOBP13、AlucOBP17的气味结合特性　出处：《吉林农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：绿盲蝽(Apolygus lucorum)是我国一种重要的农业害虫,具有灵活机动、寄主广泛和隐蔽为害等特点。化学感受行为在绿盲蝽许多生命阶段中发挥着至关重要的作用,如求偶、寄主定位和寄主转移过程中性信息素及花香类气味的探测。气味结合蛋白(odorant binding proteins,OBPs)被认为参与化学信息素感知过程中的初始生化识别阶段。随着盲蝽性信息素组分的鉴定以及盲蝽性诱剂在田间应用中的发展,干扰昆虫的化学信息通讯已经成为一种害虫防治的新策略。本研究从分子水平深入探索绿盲蝽对性信息素的识别机制,为嗅觉基因的干扰或沉默及优化性诱剂组分提供理论参考。本试验主要研究内容如下:1.通过绿盲蝽触角转录组测序共获得38个OBPs基因,包括26个“classic”OBPs和12“Plus-C”OBPs基因。系统发育树结果表明绿盲蝽OBPs蛋白与半翅目中其它盲蝽的OBPs蛋白在进化树中聚集在一起,亲缘关系表现得更为接近。荧光定量qRT-PCR结果表明在新鉴定的23个OBPs基因中,有11个OBPs基因在绿盲蝽雌雄成虫触角中的表达水平显著高于其他组织,其中有3个在雄虫触角表达水平显著高于雌虫触角,有6个在雌虫触角表达水平显著高于雄虫触角,暗示着它们可能在探测性信息素或寄主植物挥发物中发挥着作用;有3个OBPs基因在口器表达水平显著高于其他组织;有4个OBPs基因在头部表达水平显著高于其他组织;有2个OBPs基因在足部表达水平显著高于其他组织;有1个OBP基因在腹部表达水平显著高于其他组织;有1个OBP基因在翅部表达水平显著高于其他组织;有2个OBPs基因在主要组织如触角、口器、头部、足和翅中都有表达并且差异不显著。大多数具有直系同源基因的绿盲蝽OBPs基因与它们的直系同源基因有着相似的表达谱,强烈地表明这些同源基因在嗅觉或味觉中具有相似的功能。2.筛选出两个绿盲蝽male-biased触角气味结合蛋白AlucOBP13和AlucOBP17作为潜在的性信息素结合蛋白(PBPs),这两个OBPs可能与绿盲蝽识别性信息素过程息息相关。对AlucOBP13和AlucOBP17进行克隆、表达和纯化,通过荧光竞争结合试验和微量热泳动试验对它们与9种潜在的盲蝽性信息素物质,以及59种绿盲蝽偏好寄主植物挥发物、绿叶挥发物、萜烯类物质和引起绿盲蝽较强EAG反应的气味物质的结合能力进行测定。结果表明AlucOBP13结合谱较窄,只能结合β-紫罗酮,我们推测AlucOBP13可能是普通气味分子结合蛋白(GOBP),在绿盲蝽寄主定位和寄主转移过程中发挥着重要的作用;AlucOBP17能与盲蝽性信息素类似物丁酸辛酯、己酸己酯以及棉花挥发物十二醛、花香类物质β-紫罗酮有效的结合,我们推测AlucOBP17可能是潜在的PBP,并在绿盲蝽识别性信息素、寄主定位和寄主转移过程中发挥着重要的作用。
[Abstract]:A.lucorum (Apolygus lucorum) is a kind of important agricultural pests, has the characteristics of flexible, wide host and hidden damage. Chemosensory behavior plays a vital role in many a.lucorum stages of life such as mating, host location and host transfer detection process neutral pheromone and floral scent the odorant binding protein (odorant binding, proteins, OBPs) is considered the initial biochemical identification stage in chemical pheromone perception process. With the blind sex pheromone component identification and blind sex pheromone in the field of development, chemical information and communication interference in insects has become a new strategy for pest control this study. From the in-depth exploration of the molecular level l.lucorum of sex pheromone recognition mechanism for olfactory gene interference or silencing and optimization of sex pheromone components and provide a theoretical reference. This experiment mainly study The contents are as follows: 1. by a.lucorum antennal transcriptome sequencing of 38 OBPs genes were obtained, including 26 "classic" OBPs and 12 "Plus-C" OBPs gene. The phylogenetic tree showed that other Lygus lucorum OBPs protein and OBPs protein in Hemiptera in evolutionary tree together, relationship performance get closer. Fluorescence quantitative qRT-PCR results showed that the 23 OBPs gene identification, expression level of OBPs gene in 11 lucorum antennae of male and female adults was significantly higher than that of other organizations, 3 of them expressed in male antenna were significantly higher than that of female antennae, there are 6 levels of expression in female antennae was significantly higher than that in male antenna, suggesting that they may be in the detection of sex pheromone and host plant volatiles play a role in the play; with the level of expression of 3 OBPs genes in the mouthparts was significantly higher than that in other tissues; 4 OBPs gene expression level in the head There is significantly higher than that of other organizations; the expression level of OBPs gene in 2 foot was significantly higher than that in other tissues; the expression level of OBP gene is 1 in the abdomen was significantly higher than that of other organizations; with the level of expression of 1 OBP genes in the wing was significantly higher than that of other organizations; there are 2 OBPs genes in main tissues such as antennae, mouthparts, head, have and there was no significant difference between the expression of both foot and wings. Most of the orthologous orthologs lucorum OBPs gene and they have a similar expression profile strongly suggests that these homologous genes with similar function as.2. screened two lucorum male-biased antenna odorant binding protein AlucOBP13 and AlucOBP17 as potential pheromone binding protein in smell or taste in (PBPs), the two OBPs may be closely related to a.lucorum recognition of sex pheromone. Cloning of AlucOBP13 and AlucOBP17, expression and purification, By fluorescence binding assay and microcalorimetry swimming test for them and 9 potential bug sex pheromone substances, as well as 59 kinds of a.lucorum preference of host plant volatiles, green leaf volatiles, combined with the ability of terpenes and caused strong reaction of a.lucorum EAG odorants were determined. The results showed that AlucOBP13 combined with narrow spectrum, only with betalonone, we speculate that AlucOBP13 may be a general odorant binding protein (GOBP), plays an important role in the transfer of a.lucorum host location and host in the process; AlucOBP17 and fleahopper sex pheromone analogs octyl butyrate, Hexyl Hexanoate and cotton volatiles twelve aldehyde, floral material with betalonone effectively, we speculate that AlucOBP17 may be a potential PBP, and in lucorum recognition of sex pheromone, host location and host transfer process plays an important role.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433

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