本文關鍵詞： 豆野螟 MvitGOBP1 MvitCSP1 MvitOBP1 基因克隆 競爭結合特性 三維結構模擬 分子對接　出處：《華中師范大學》2016年碩士論文　論文類型：學位論文

【摘要】：在漫長的進化歷程中,昆蟲逐漸具備了極其靈敏的嗅覺系統(tǒng),并以此感知來自于外界的多種化學刺激并作出相應的生理行為反應,如尋找配偶、定位食物源和棲息地,兩性交流,聚集產卵和趨避敵害等,由此可見,嗅覺及化學感受系統(tǒng)在昆蟲的各項生命活動中發(fā)揮著重要功能。通過鑒定篩選靶標害蟲信息素成分干擾和阻斷昆蟲的嗅覺識別行為,并將其成功應用于田間實踐一直是害蟲綜合生物防控的重要措施之一。豆野螟是一種在世界范圍內都具有較大危害的豆科作物害蟲,其幼蟲具有較強的鉆蛀習性,化學藥劑的大量使用不易殺死害蟲,并容易導致農藥殘留超標,嚴重威脅各蔬菜基地豆類蔬菜產品的供應和食品安全。豆野螟主要分布在熱帶和亞熱帶地區(qū),幼蟲主要蛀食豆科作物幼嫩的花、葉和豆莢等,給豆科蔬菜生產造成了巨大的損失�；谡n題組前期豆野螟觸角轉錄組測序結果,我們克隆、表達并純化了豆野螟MvitGOBP1、MvitCSP1、MvitOBP1,采用RT-PCR技術分析檢測這三個基因在豆野螟成蟲的不同組織的表達情況。利用熒光競爭結合實驗分析研究了MvitGOBP1、 MvitCSP1、MvitOBP1與寄主植物花揮發(fā)性化合物的結合,并利用三維結構模擬以及分子對接技術對MvitGOBP1、MvitCSP1蛋白與配體分子的可能的關鍵結合位點進行預測。主要研究結果如下：1.豆野螟MvitGOBP1、MvitCSP1、MvitOBP1的克隆、序列和進化分析克隆獲得豆野螟三個嗅覺基因,分別命名為MvitGOBP1、MvitCSP1、MvitOBP1,序列分析三個基因的ORF分別為486bp、387bp和471bp。多重序列比對顯示,MvitGOBP1、MvitCSP1和MvitOBP1均與鱗翅目其他昆蟲的相應基因具有較高的相似性。系統(tǒng)進化分析發(fā)現(xiàn),MvitGOBP1與CmedGOBP1聚為一支,MvitCSP1和CmedCSP,MvitOBP1和CmedOBP11分別具有較高序列相似性。2.組織特異性表達分析利用熒光定量PCR技術分析檢測了MvitGOBP1、MvitCSPl和MvitOBP1在豆野螟成蟲不同組織的表達水平。結果表明,這三個基因主要在觸角中高表達,在頭、足和翅等組織中分別有少量的表達分布。3. MvitGOBP1、MvitCSP1和MvitOBP1的原核表達及親和層析純化將構建好的原核表達重組載體轉入到大腸桿菌BL21感受態(tài)細胞中,不同IPTG條件誘導后破碎取上清,采用Ni柱親和層析的方法來純化目的蛋白,并利用腸激酶切除His標簽。SDS-PAGE分析檢測顯示,這三個蛋白大小在25 kD到35kD之間產生特異性的條帶。4.熒光競爭結合實驗熒光競爭結合實驗結果顯示,MvitGOBP1對1H-Indol-4-ol(4-羥基吲哚)、Butanoic acid butyl ester(丁酸丁酯)和Limonene(檸檬烯)的結合能力較強;MvitCSP1對1H-Indol-4-ol和2-methyl-3-phenylpropanal(2-甲基-3-苯基丙醛)的結合能力較強；MvitOBP1對1H-Indol-4-ol和Butanoic acid butyl ester的結合能力較強。5.田間誘捕試驗基于嗅覺目的蛋白與寄主揮發(fā)物的結合特性分析結果,我們從17種豇豆花揮發(fā)物氣味分子中選取了六種結合能力較強的化合物來進行田間誘捕實驗,分別是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)。田間誘捕實驗結果顯示,相對于空白對照組,這六種化合物均能夠有效吸引雌蛾,產生類似產卵聚集效應,其中檸檬烯的誘捕效果最為顯著。6.同源建模和分子對接通過構建目的蛋白三維結構模型與分子對接分析,我們發(fā)現(xiàn)MvitGOBP1和MvitCSP1的結合腔主要由疏水性氨基酸組成,促進兩種嗅覺蛋白與寄主揮發(fā)物成分1H-indol-4-ol和2-methyl-3-phenylpropanal的特異性結合,推測可能的關鍵氨基酸位點分別是Asn83和Arg127,Thr26和Glu63。上述試驗結果表明,MvitGOBP1、MvitCSP1和MvitOBP1在豆野螟不同組織中分布廣泛,結合特性分析和田間誘捕試驗表明,MvitGOBP1、MvitCSP1和MvitOBP1在豆野螟的寄主識別以及產卵定位等嗅覺生理活動中發(fā)揮著重要作用。
[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.

【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S433

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