苹果蠹蛾信息素结合蛋白的研究及活性气味分子的筛选

发布时间：2018-06-27 00:37

  本文选题：苹果蠹蛾 + 气味分子　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：苹果蠹蛾作为一种全球性的果树害虫,每年都会对水果产业造成巨大损失。关于利用气味分子来调节苹果蠹蛾的行为从而达到防治的目的,目前虽然也有诸多研究,但都收效甚微,只发现了寥寥数种对苹果蠹蛾有诱集效果的气味分子,已经商品化的仅有苹果蠹蛾性信息素Codlemone一种。传统的气味分子筛选手段主要依赖于昆虫对气味分子的行为反应,不仅费时费力,还严重制约了气味分子的筛选范围。参照新药研发的策略,有学者提出了“Reverse Chemical Ecology”的概念,主要是以气味分子与嗅觉相关蛋白的亲和度来筛选对昆虫具有潜在生理活性的气味分子。这一概念的核心是气味分子与蛋白的相互作用,该部分可通过分子模拟来实现,主要手段是基于嗅觉相关蛋白结构模型的气味分子高通量筛。这一方法能加快气味分子的筛选过程,大批量的筛选出对苹果蠹蛾有生理活性的气味分子。在昆虫的嗅觉相关蛋白中,气味结合蛋白(OBPs,Odorant Binding Proteins)是研究最透彻的,适合用于气味分子的筛选。本研究基于“Reverse Chemical Ecology”的理念,在分析苹果蠹蛾2种信息素结合蛋白CpomPBP1(Pheromone Binding Protein 1)和CpomPBP2(Pheromone Binding Protein 2)的生化性质和功能的基础上,通过分子模拟,建立了有效筛选气味分子的新方法,并对这2种苹果蠹蛾信息素结合蛋白与气味分子作用的关键氨基酸及关键相互作用进行了分析。主要研究成果如下:1.苹果蠹蛾CpomPBP2的功能及其与气味分子的结合模式研究构建了一个由31种备选化合物组成的气味分子集合,主要包括苹果蠹蛾性腺提取物以及寄主植物苹果和梨的挥发物。基于虚拟筛选和结合实验,建立了一种可以快速筛选气味分子的方法,虚拟筛选与实验结果的一致性,验证了这种方法的可靠性。为了更好的指导气味分子筛选,我们还对CpomPBP2与气味分子之间的结合模式及结合自由能进行了分析,揭示了参与CpomPBP2与气味分子互作过程的氨基酸,并通过丙氨酸突变扫描技术进行了验证。此外,结合模式和结合自由能分析结果还显示,氢键和疏水作用是CpomPBP2与气味分子的关键相互作用。能对这两种作用方式造成干扰的因素如能形成氢键的官能团以及气味分子碳链的长短等,均会影响CpomPBP2与气味分子的亲和度。在此基础上,我们对CpomPBP2 C-端的功能进行了研究。通过比较不同pH条件下CpomPBP2和TPBP2(去掉C-端的CpomPBP2)与气味分子的结合能力可发现,C-端不仅在低pH条件下阻碍CpomPBP2与气味分子的结合,还影响气味分子的上载过程(uploading process)。2.苹果蠹蛾CpomPBP1的克隆表达及功能分析采用简并PCR和RACE相结合的手段,克隆得到了CpomPBP1的ORF序列,对其进行基因和氨基酸序列分析,可发现该蛋白具有昆虫OBPs的典型特征。通过RT-PCR,Western blot和触角免疫荧光检测等技术从mRNA和蛋白水平上对CpomPBP1的组织分布进行了描述,发现CpomPBP1属于触角特异性蛋白。使用重组CpomPBP1进行竞争结合实验,发现了3种可与CpomPBP1结合的气味分子,且这3种气味分子均在分子结构上具有一定的相似性,这也体现了昆虫嗅觉系统的特异性。对CpomPBP1的C-端功能进行深入研究可发现,C-端去除后得到的TPBP1,其结合能力虽然没变化,与气味分子的结合却变得对pH不敏感,表明C-端在低pH条件下会阻碍气味分子与蛋白的结合。综合内源荧光检测的结果可以推测,pH会引起CpomPBP1的构象变化,且这种变化可能主要体现在C-端上。3.CpomPBP1与信息素Codlemone作用的关键氨基酸位点研究采用同源模建和分子对接的方法构建了CpomPBP1以及CpomPBP1/Codlemone复合物的三维结构图,并通过分子动力学以及结合自由能计算对该复合物的稳定性进行了描述。通过对结合自由能能量分项的分析确认了Codlemone结合CpomPBP1的主要能量驱动力为范德瓦斯相互作用和静电相互作用,而Codlemone的-OH与Trp37形成的氢键作用是稳定CpomPBP1/Codlemone复合物的主要贡献者。通过对各氨基酸残基的总能量贡献和侧链能量贡献进行分解,最终选取了侧链能量贡献大于0.5kcal/mol的Phe12、Phe36、Trp37、Ile52、Ile94和Phe118等6个残基位点进行丙氨酸突变扫描和生物学定点突变,最终确认了Phe12和Trp37为CpomPBP1结合Codlemone的关键位点。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433.4
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本文编号：2072012