本文選題：蘋果蠹蛾 + 氣味分子�。� 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：蘋果蠹蛾作為一種全球性的果樹害蟲,每年都會對水果產(chǎn)業(yè)造成巨大損失。關(guān)于利用氣味分子來調(diào)節(jié)蘋果蠹蛾的行為從而達到防治的目的,目前雖然也有諸多研究,但都收效甚微,只發(fā)現(xiàn)了寥寥數(shù)種對蘋果蠹蛾有誘集效果的氣味分子,已經(jīng)商品化的僅有蘋果蠹蛾性信息素Codlemone一種。傳統(tǒng)的氣味分子篩選手段主要依賴于昆蟲對氣味分子的行為反應(yīng),不僅費時費力,還嚴重制約了氣味分子的篩選范圍。參照新藥研發(fā)的策略,有學(xué)者提出了“Reverse Chemical Ecology”的概念,主要是以氣味分子與嗅覺相關(guān)蛋白的親和度來篩選對昆蟲具有潛在生理活性的氣味分子。這一概念的核心是氣味分子與蛋白的相互作用,該部分可通過分子模擬來實現(xiàn),主要手段是基于嗅覺相關(guān)蛋白結(jié)構(gòu)模型的氣味分子高通量篩。這一方法能加快氣味分子的篩選過程,大批量的篩選出對蘋果蠹蛾有生理活性的氣味分子。在昆蟲的嗅覺相關(guān)蛋白中,氣味結(jié)合蛋白(OBPs,Odorant Binding Proteins)是研究最透徹的,適合用于氣味分子的篩選。本研究基于“Reverse Chemical Ecology”的理念,在分析蘋果蠹蛾2種信息素結(jié)合蛋白CpomPBP1(Pheromone Binding Protein 1)和CpomPBP2(Pheromone Binding Protein 2)的生化性質(zhì)和功能的基礎(chǔ)上,通過分子模擬,建立了有效篩選氣味分子的新方法,并對這2種蘋果蠹蛾信息素結(jié)合蛋白與氣味分子作用的關(guān)鍵氨基酸及關(guān)鍵相互作用進行了分析。主要研究成果如下:1.蘋果蠹蛾CpomPBP2的功能及其與氣味分子的結(jié)合模式研究構(gòu)建了一個由31種備選化合物組成的氣味分子集合,主要包括蘋果蠹蛾性腺提取物以及寄主植物蘋果和梨的揮發(fā)物�；谔摂M篩選和結(jié)合實驗,建立了一種可以快速篩選氣味分子的方法,虛擬篩選與實驗結(jié)果的一致性,驗證了這種方法的可靠性。為了更好的指導(dǎo)氣味分子篩選,我們還對CpomPBP2與氣味分子之間的結(jié)合模式及結(jié)合自由能進行了分析,揭示了參與CpomPBP2與氣味分子互作過程的氨基酸,并通過丙氨酸突變掃描技術(shù)進行了驗證。此外,結(jié)合模式和結(jié)合自由能分析結(jié)果還顯示,氫鍵和疏水作用是CpomPBP2與氣味分子的關(guān)鍵相互作用。能對這兩種作用方式造成干擾的因素如能形成氫鍵的官能團以及氣味分子碳鏈的長短等,均會影響CpomPBP2與氣味分子的親和度。在此基礎(chǔ)上,我們對CpomPBP2 C-端的功能進行了研究。通過比較不同pH條件下CpomPBP2和TPBP2(去掉C-端的CpomPBP2)與氣味分子的結(jié)合能力可發(fā)現(xiàn),C-端不僅在低pH條件下阻礙CpomPBP2與氣味分子的結(jié)合,還影響氣味分子的上載過程(uploading process)。2.蘋果蠹蛾CpomPBP1的克隆表達及功能分析采用簡并PCR和RACE相結(jié)合的手段,克隆得到了CpomPBP1的ORF序列,對其進行基因和氨基酸序列分析,可發(fā)現(xiàn)該蛋白具有昆蟲OBPs的典型特征。通過RT-PCR,Western blot和觸角免疫熒光檢測等技術(shù)從mRNA和蛋白水平上對CpomPBP1的組織分布進行了描述,發(fā)現(xiàn)CpomPBP1屬于觸角特異性蛋白。使用重組CpomPBP1進行競爭結(jié)合實驗,發(fā)現(xiàn)了3種可與CpomPBP1結(jié)合的氣味分子,且這3種氣味分子均在分子結(jié)構(gòu)上具有一定的相似性,這也體現(xiàn)了昆蟲嗅覺系統(tǒng)的特異性。對CpomPBP1的C-端功能進行深入研究可發(fā)現(xiàn),C-端去除后得到的TPBP1,其結(jié)合能力雖然沒變化,與氣味分子的結(jié)合卻變得對pH不敏感,表明C-端在低pH條件下會阻礙氣味分子與蛋白的結(jié)合。綜合內(nèi)源熒光檢測的結(jié)果可以推測,pH會引起CpomPBP1的構(gòu)象變化,且這種變化可能主要體現(xiàn)在C-端上。3.CpomPBP1與信息素Codlemone作用的關(guān)鍵氨基酸位點研究采用同源模建和分子對接的方法構(gòu)建了CpomPBP1以及CpomPBP1/Codlemone復(fù)合物的三維結(jié)構(gòu)圖,并通過分子動力學(xué)以及結(jié)合自由能計算對該復(fù)合物的穩(wěn)定性進行了描述。通過對結(jié)合自由能能量分項的分析確認了Codlemone結(jié)合CpomPBP1的主要能量驅(qū)動力為范德瓦斯相互作用和靜電相互作用,而Codlemone的-OH與Trp37形成的氫鍵作用是穩(wěn)定CpomPBP1/Codlemone復(fù)合物的主要貢獻者。通過對各氨基酸殘基的總能量貢獻和側(cè)鏈能量貢獻進行分解,最終選取了側(cè)鏈能量貢獻大于0.5kcal/mol的Phe12、Phe36、Trp37、Ile52、Ile94和Phe118等6個殘基位點進行丙氨酸突變掃描和生物學(xué)定點突變,最終確認了Phe12和Trp37為CpomPBP1結(jié)合Codlemone的關(guān)鍵位點。
[Abstract]:As a global pest of fruit tree, the pinetmoth moth has caused great losses to the fruit industry every year. There are many studies on the use of odorants to regulate the behavior of the moth moth, but there are few effects. Only a few odorants have been found to induce the effect of the moth moth. It has been commercialized only a kind of sex pheromone Codlemone of the moth moth. The traditional screening method of odorants is mainly dependent on the behavior of insect to odor molecules. It is not only time-consuming and laborious, but also severely restricts the screening range of odorant molecules. According to the strategy of new drug research and development, the concept of "Reverse Chemical Ecology" is put forward by the scholars. The core of this concept is the interaction between odorant molecules and proteins, which can be realized by molecular simulation. The main means are high throughput screening of odorant molecules based on the olfactory phase protein structure model. One method can accelerate the screening process of odorants and large batch screening of odorant molecules that have physiological activity to the moth moth. In the insect's olfactory related protein, OBPs (Odorant Binding Proteins) is the most thorough research and suitable for the screening of odorant molecules. This study is based on "Reverse Chemical Ecology". On the basis of the analysis of the biochemical properties and functions of 2 pheromone binding proteins CpomPBP1 (Pheromone Binding Protein 1) and CpomPBP2 (Pheromone Binding Protein 2) of the moth moth, a new method for effective screening of odor molecules was established by molecular simulation, and the pheromone binding proteins and odorants of the 2 species of moth moth were made. The key amino acids and key interactions were analyzed. The main research results were as follows: 1. the function of the CpomPBP2 and the combination mode of the odorant molecules were used to construct a collection of odor molecules composed of 31 kinds of alternative compounds, mainly including the sex gland extracts of the moth moth and the volatiles of the host plants, apples and pears. Based on the virtual screening and experiment, a method of rapid screening of odorant molecules was established. The consistency between the virtual screening and the experimental results proved the reliability of the method. In order to better guide the screening of odor molecules, we also analyzed the combination mode and the free energy of the CpomPBP2 and the odorant molecules. The amino acids involved in the interaction between CpomPBP2 and odorant molecules were revealed and verified by the alanine mutation scanning technique. Furthermore, the binding mode and binding free energy analysis showed that the hydrogen bond and hydrophobicity were the key interactions between CpomPBP2 and odorant molecules. The factors that could cause interference to these two modes of action could be considered as possible. On the basis of this, we have studied the function of the CpomPBP2 C- end, and on this basis, we have studied the function of the CpomPBP2 C- end. By comparing the binding ability of CpomPBP2 and TPBP2 (CpomPBP2 removed to the C- end) with the odorant molecules under different pH conditions, the C- end is not only low. The combination of CpomPBP2 and odorant molecules was hindered by pH, and the cloning expression and function analysis of the odorant (uploading process).2. moth CpomPBP1 were cloned and the ORF sequence of CpomPBP1 was cloned. The gene and amino acid sequence of the CpomPBP1 were cloned and the protein was found. There are typical characteristics of the insect OBPs. The tissue distribution of CpomPBP1 is described from mRNA and protein levels by RT-PCR, Western blot and antennae immunofluorescence. It is found that CpomPBP1 belongs to the antennae specific protein. 3 kinds of odorant molecules, which can be combined with CpomPBP1, are identified by the competition binding experiment with the recombinant CpomPBP1. 3 kinds of odors have some similarity on the molecular structure, which also embodies the specificity of the insect olfactory system. A thorough study of the C- terminal function of the CpomPBP1 can be found that the TPBP1 which is obtained after the C- end is removed is not sensitive to the combination of the odorant molecules, but it is not sensitive to the pH, indicating that the C- end is under the low pH condition. The combination of odorant molecules and proteins can be hindered. The results of integrated endogenous fluorescence detection can speculate that pH will cause conformation changes of CpomPBP1, and this change may be mainly reflected in the key amino acid sites of the.3.CpomPBP1 and pheromone Codlemone on the C- end and the construction of CpomPBP1 and C by homologous modeling and molecular docking. The three-dimensional structure of the pomPBP1/Codlemone complex is described and the stability of the complex is described by the molecular dynamics and the calculation of the free energy. The main energy driving force of the Codlemone combined with CpomPBP1 is confirmed by the analysis of the free energy energy separation. The interaction of van der WAAS and the electrostatic interaction, and the Codlemo The hydrogen bond formed by -OH and Trp37 of NE is the main contributor to the stable CpomPBP1/Codlemone complex. By decomposing the total energy contribution of the amino acid residues and the contribution of the side chain energy, the 6 residues of Phe12, Phe36, Trp37, Ile52, Ile94 and Phe118 are selected for the alanine process of the side chain energy contribution more than 0.5kcal/mol. Variable scanning and site directed mutagenesis confirmed that Phe12 and Trp37 were the key sites for CpomPBP1 binding to Codlemone.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S433.4
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本文編號：2072012