豆野螟嗅觉相关基因GOBP1、CSP1和OBP1的功能分析

发布时间：2018-01-26 05:20

本文关键词： 豆野螟 MvitGOBP1 MvitCSP1 MvitOBP1 基因克隆竞争结合特性三维结构模拟分子对接　出处：《华中师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：在漫长的进化历程中,昆虫逐渐具备了极其灵敏的嗅觉系统,并以此感知来自于外界的多种化学刺激并作出相应的生理行为反应,如寻找配偶、定位食物源和栖息地,两性交流,聚集产卵和趋避敌害等,由此可见,嗅觉及化学感受系统在昆虫的各项生命活动中发挥着重要功能。通过鉴定筛选靶标害虫信息素成分干扰和阻断昆虫的嗅觉识别行为,并将其成功应用于田间实践一直是害虫综合生物防控的重要措施之一。豆野螟是一种在世界范围内都具有较大危害的豆科作物害虫,其幼虫具有较强的钻蛀习性,化学药剂的大量使用不易杀死害虫,并容易导致农药残留超标,严重威胁各蔬菜基地豆类蔬菜产品的供应和食品安全。豆野螟主要分布在热带和亚热带地区,幼虫主要蛀食豆科作物幼嫩的花、叶和豆荚等,给豆科蔬菜生产造成了巨大的损失。基于课题组前期豆野螟触角转录组测序结果,我们克隆、表达并纯化了豆野螟MvitGOBP1、MvitCSP1、MvitOBP1,采用RT-PCR技术分析检测这三个基因在豆野螟成虫的不同组织的表达情况。利用荧光竞争结合实验分析研究了MvitGOBP1、 MvitCSP1、MvitOBP1与寄主植物花挥发性化合物的结合,并利用三维结构模拟以及分子对接技术对MvitGOBP1、MvitCSP1蛋白与配体分子的可能的关键结合位点进行预测。主要研究结果如下：1.豆野螟MvitGOBP1、MvitCSP1、MvitOBP1的克隆、序列和进化分析克隆获得豆野螟三个嗅觉基因,分别命名为MvitGOBP1、MvitCSP1、MvitOBP1,序列分析三个基因的ORF分别为486bp、387bp和471bp。多重序列比对显示,MvitGOBP1、MvitCSP1和MvitOBP1均与鳞翅目其他昆虫的相应基因具有较高的相似性。系统进化分析发现,MvitGOBP1与CmedGOBP1聚为一支,MvitCSP1和CmedCSP,MvitOBP1和CmedOBP11分别具有较高序列相似性。2.组织特异性表达分析利用荧光定量PCR技术分析检测了MvitGOBP1、MvitCSPl和MvitOBP1在豆野螟成虫不同组织的表达水平。结果表明,这三个基因主要在触角中高表达,在头、足和翅等组织中分别有少量的表达分布。3. MvitGOBP1、MvitCSP1和MvitOBP1的原核表达及亲和层析纯化将构建好的原核表达重组载体转入到大肠杆菌BL21感受态细胞中,不同IPTG条件诱导后破碎取上清,采用Ni柱亲和层析的方法来纯化目的蛋白,并利用肠激酶切除His标签。SDS-PAGE分析检测显示,这三个蛋白大小在25 kD到35kD之间产生特异性的条带。4.荧光竞争结合实验荧光竞争结合实验结果显示,MvitGOBP1对1H-Indol-4-ol(4-羟基吲哚)、Butanoic acid butyl ester(丁酸丁酯)和Limonene(柠檬烯)的结合能力较强;MvitCSP1对1H-Indol-4-ol和2-methyl-3-phenylpropanal(2-甲基-3-苯基丙醛)的结合能力较强；MvitOBP1对1H-Indol-4-ol和Butanoic acid butyl ester的结合能力较强。5.田间诱捕试验基于嗅觉目的蛋白与寄主挥发物的结合特性分析结果,我们从17种豇豆花挥发物气味分子中选取了六种结合能力较强的化合物来进行田间诱捕实验,分别是Butanoic acid butyl ester、Limonene、1H-Indol-4-ol、2-methyl-3-phenylpropanal、 Butanoic acid octyl ester(丁酸辛酯)和4-ethylpropiophenone(乙基苯丙酮)。田间诱捕实验结果显示,相对于空白对照组,这六种化合物均能够有效吸引雌蛾,产生类似产卵聚集效应,其中柠檬烯的诱捕效果最为显著。6.同源建模和分子对接通过构建目的蛋白三维结构模型与分子对接分析,我们发现MvitGOBP1和MvitCSP1的结合腔主要由疏水性氨基酸组成,促进两种嗅觉蛋白与寄主挥发物成分1H-indol-4-ol和2-methyl-3-phenylpropanal的特异性结合,推测可能的关键氨基酸位点分别是Asn83和Arg127,Thr26和Glu63。上述试验结果表明,MvitGOBP1、MvitCSP1和MvitOBP1在豆野螟不同组织中分布广泛,结合特性分析和田间诱捕试验表明,MvitGOBP1、MvitCSP1和MvitOBP1在豆野螟的寄主识别以及产卵定位等嗅觉生理活动中发挥着重要作用。
[Abstract]:In the long course of evolution, gradually with the insect olfactory system is extremely sensitive, and this perception from the outside a variety of chemical stimulation and make physiological response, such as looking for a spouse, locating food source and habitat, sexual communication, and averse predators gather to spawn, therefore, olfactory and chemosensory systems play the important function in the life activities of the insects. Screening target insect pheromone components interference and blocking the olfactory behavior of insects through the identification, and successfully applied in the field of practice has been one of the important measures of integrated pest biological control. Maruca vitrata is a great harm in the world within the scope of leguminous crops the larvae borer pests, has strong chemical habit, extensive use is not easy to kill pests, and easily lead to exceed the standard of pesticide residues, a serious threat to the vegetable base The supply of food safety and legumes vegetable products. Maruca vitrata mainly distributed in tropical and subtropical regions, the larvae mainly eats tender legume flowers, leaves and pods, caused huge losses to vegetable production. Legume ourprevious Maruca vitrata antennal transcriptome sequencing results, based on our cloning, expression and Maruca vitrata MvitGOBP1, purified MvitCSP1, MvitOBP1 analysis, to detect the expression of these three genes in different tissues of adult Maruca vitrata using RT-PCR technology. Using fluorescence competition of MvitGOBP1, MvitCSP1 combined with the experimental analysis, the combination of MvitOBP1 and host plant flowers of volatile compounds, and the use of three-dimensional structure modeling and molecular docking technology the key of MvitGOBP1, MvitCSP1 protein and ligand molecules possible binding site prediction. The main results are as follows: 1. Maruca vitrata MvitGOBP1, MvitCSP1, MvitOBP1 cloning The sequence and phylogenetic analysis, cloned Maruca vitrata three olfactory genes, named MvitGOBP1, MvitCSP1, MvitOBP1, sequence analysis of three genes of ORF were 486bp, 387bp and 471bp. multiple sequence alignment showed that MvitGOBP1, MvitCSP1 and MvitOBP1 corresponding genes were Lepidoptera insects and other high similarity system. Phylogenetic analysis showed that MvitGOBP1 and CmedGOBP1 were clustered into a, MvitCSP1 and CmedCSP, MvitOBP1 and CmedOBP11 respectively with high sequence similarity of.2. tissue specific expression analysis using fluorescence quantitative PCR detection and analysis of MvitGOBP1, the expression level of MvitCSPl and MvitOBP1 in different tissues of adult Maruca vitrata. The results showed that the three genes are high in the expression, the tentacles on the head, legs and wings and other tissues respectively have a small amount of expression and distribution of.3. MvitGOBP1, MvitCSP1 and MvitOBP1 of the prokaryotic expression and affinity chromatography purification Construction of the Recombinant Prokaryotic expression vector into E.coli BL21 competent cells, induced by different IPTG conditions after crushing the supernatant, methods using Ni affinity chromatography to purify the protein, and the use of enterokinase to remove His tag.SDS-PAGE analysis showed that the three protein in size from 25 kD to.4. with fluorescent competition according to the experimental result in specific 35kD fluorescence competition between a combination of experimental results show that the MvitGOBP1 of 1H-Indol-4-ol (4- Butanoic acid butyl hydroxyindole), ester (butyl butyrate) and Limonene (limonene) the strong binding capacity of 1H-Indol-4-ol and MvitCSP1; 2-methyl-3-phenylpropanal (2- methyl -3- phenyl aldehyde) the strong binding capacity of 1H-Indol-4-ol MvitOBP1; Butanoic acid and butyl ester.5. of the strong binding capacity field trapping test analysis combined with the characteristics of olfactory protein and host volatiles based on The results, we spend volatile odorants from 17 cowpea selected six kinds of field trapping experiments combined with compound ability, are Butanoic acid butyl ester, Limonene, 1H-Indol-4-ol, 2-methyl-3-phenylpropanal, Butanoic acid octyl ester (D suanxin ester) and 4-Ethylpropiophenone (ethyl benzene acetone). Field trapping experiments show that compared with the control group, the six compounds are able to effectively attract female moths, produce similar spawning aggregation effect, the limonene trapping effect is the most significant.6. homology modeling and molecular docking and molecular 3D structure by constructing a model protein docking analysis, we found that combining cavity MvitGOBP1 and MvitCSP1 is mainly composed of hydrophobic amino acids, to promote the two kinds of specific olfactory proteins and host volatile components of 1H-indol-4-ol and 2-methyl-3-phenylpropanal combination, That may be the key amino acids were Asn83 and Arg127, Thr26 and Glu63., the test results show that MvitGOBP1, MvitCSP1 and MvitOBP1 are widely distributed in different tissues of Maruca vitrata, combined with the characteristics of analysis and field trap test showed that MvitGOBP1, MvitCSP1 and MvitOBP1 play an important role in the physiological activities of the host recognition of Maruca vitrata and spawning location of smell.

【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433

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