云斑天牛Minus-C OBPs与配基的结合与释放机制
发布时间：2018-08-12 08:32
【摘要】：云斑天牛Batocera horsfieldi(Hope),又名云斑白条天牛,在我国分布广泛,是林业上重要的蛀干害虫,其灵敏的嗅觉系统在寄主定位、交配和产卵等方面发挥着重要的作用。而昆虫的气味结合蛋白(OBPs)可以携带环境中的疏水性气味分子到达气味受体,在特异性识别信息化学物质的过程中起着重要的功能。若能明确云斑天牛气味结合蛋白与配体分子的结合与释放机制,可为其行为调节剂的研究提供重要的理论依据。为此,本课题以云斑天牛为研究对象,测定了Bhor OBPs与17种寄主挥发物的荧光竞争结合特性,并利用计算机进行同源模建和分子对接,配合氨基酸定点突变技术,分析了Bhor OBPm2的结构特点及其与配体分子的结合与释放机制。主要研究结果如下:1.云斑天牛Minus-C OBPs基因克隆、蛋白表达和纯化成功构建重组质粒p GEX-6P-1-Bhor OBPm1,p ET30a-Bhor OBPm2和p ET17bBhor OBPm4。p GEX-6P-1-Bhor OBPm1在上清中表达,通过GST 4B亲和层析柱获得Bhor OBPm1的纯蛋白;p ET30a-Bhor OBPm2和p ET17b-Bhor OBPm4在包涵体中表达,分别通过Ni离子亲和层析柱及DE52弱阴离子交换柱在体外成功获得Bhor OBPm2和Bhor OBPm4的纯蛋白。2.云斑天牛Minus-C OBPs结合特性分析以1-NPN为荧光探针分别测定了云斑天牛3个Minus-C OBPs与寄主挥发物在酸性和中性环境下的结合能力。Bhor OBPm1在中性环境下与水杨醛有较弱的结合能力,在酸性环境下与马鞭草烯酮有较强结合能力;Bhor OBPm2在中性环境与己醛、反-2-己烯醛以及反-2-己烯醇有较强的结合能力,在酸性条件下仅与反-2-己烯醇、己醛和2-甲基丁醛表现出较弱的结合能力;Bhor OBPm4在中性环境下与所有挥发物都无结合能力,在酸性环境下,仅与柠檬烯氧化物、水杨醛和顺-3-己烯醇表现出较弱的结合能力。3.云斑天牛Bhor OBPm2同源建模与分子对接以同源蛋白Agam OBP1(2ERB)为模板,建模得到Bhor OBPm2的三维结构,其具有六个α螺旋,两个二硫键,属于Minus-C OBPs家族。与模板蛋白Agam OBP1(2ERB)的C端结构相似,Bhor OBPm2的C末端的氨基酸残基Phe123与Tyr50形成氢键,将C端固定在结合腔的表面。Bhor OBPm2与56种气味物质对接结果显示,六个α螺旋在Bhor OBPm2的中心形成了一个疏水性的结合腔,且在结合腔的表面形成了两个大小不同的开口。结合腔的表面大部分为疏水性区域,配体小分子主要以疏水作用力固定在腔内,而C端氨基酸残基Ile121和Phe123形成了结合腔表面唯一的极性区域。4.Bhor OBPm2的氨基酸定点突变及结合验证荧光竞争结合试验结果表明,Bhor OBPm2特异性选择链状配基,且基于碳链的长度进行选择;Bhor OBPm2与配基的结合能力受p H的影响,中性环境下的结合能力明显强于酸性环境。将Bhor OBPm2与其他OBPs进行比较,我们发现属于Minus-C OBPs家族的Bhor OBPm2与属于Classical OBPs家族的Agam OBP1、Cqui OBP1和Aaeg OBP1具有相似的结合腔结构以及配基选择取向,但由于缺失一个二硫键,Bhor OBPm2拥有更大的结合腔和更广泛的配基选择。因此我们猜测Bhor OBPm2可能是OBPs进化过程中的中间体结构。为探究Bhor OBPm2 C端的作用,我们设计了两个Bhor OBPm2的突变体C-ter113和Y50F。通过对突变体蛋白的研究发现,Bhor OBPm2的C端在配基释放的过程中并非如前人报道的起到了“盖子”作用。
[Abstract]:Batocera horsfieldi (Hope), also known as Batocera horsfieldi (Hope), is widely distributed in China and is an important stem-borer in forestry. Its sensitive olfactory system plays an important role in host location, mating and oviposition. The odor-binding proteins (OBPs) of insects can carry hydrophobic odor molecules into the atmosphere. Taste receptors play an important role in the specific recognition of informational substances. If the binding and release mechanism of odor-binding proteins and ligand molecules can be clarified, it will provide an important theoretical basis for the study of behavioral regulators. The fluorescence competitive binding characteristics of the main volatiles were studied by computer homology modeling and molecular docking. The structure characteristics of Bhor OBPm2 and its binding and release mechanism with ligand molecules were analyzed by amino acid site-directed mutagenesis technique. The main results were as follows: 1. Cloning of Minus-C OBPs gene, protein expression and purification were successful. The recombinant plasmids P GEX-6P-1-Bhor OBPm1, P ET30a-Bhor OBPm2 and pET17bhor OBPm4.p GEX-6P-1-Bhor OBPm1 were constructed and expressed in the supernatant. The purified protein of Bhor OBPm1 was obtained by GST 4B affinity chromatography. The expression of P ET30a-Bhor OBPm2 and P ET17b-Bhor OBPm4 in inclusion bodies was detected by Ni ion affinity chromatography and DE52 weak anion exchange column, respectively. Bhor OBPm 2 and Bhor OBPm4 were successfully obtained in vitro. 2. The binding properties of three Minus-C OBPs from A. yunnanensis to host volatiles in acidic and neutral environments were determined by using 1-NPN as fluorescent probe. Bhor OBPm1 had weak binding ability to salicylaldehyde in neutral environments, and in acidic and neutral environments. Bhor OBPm2 has strong binding ability with hexanal, trans-2-hexenal and trans-2-hexenol in neutral environment, but weak binding ability with trans-2-hexenol, hexanal and 2-methylbutyraldehyde in acidic environment; Bhor OBPm4 has no binding ability with all volatiles in neutral environment. The binding ability of Bhor OBPm2 was weaker than that of citrone oxides, salicylaldehyde and cis-3-hexenol in acidic environment. 3. Homologous modeling and molecular docking of Bhor OBPm2 were performed using homologous protein Agam OBP1 (2ERB) as template, and the three-dimensional structure of Bhor OBPm2 was modeled. It has six alpha helixes and two disulfide bonds, belonging to Minus-C OBPs. Similar to the C-terminal structure of the template protein Agam OBP1 (2ERB), the amino acid residue Phe123 at the C-terminal of Bhor OBPm2 forms a hydrogen bond with Tyr50 and fixes the C-terminal on the surface of the binding cavity. The docking results of Bhor OBPm2 with 56 odor compounds show that six alpha-helixes form a hydrophobic binding cavity in the center of Bhor OBPm2 and are in the binding cavity. The surface of the binding cavity is mostly hydrophobic, and the small ligand molecules are mainly fixed in the cavity by hydrophobic force. The C-terminal amino acid residues Ile121 and Phe123 form the unique polar region on the surface of the binding cavity. 4. Site-directed mutagenesis of amino acids in Bhor OBPm2 and binding verification fluorescence competitive binding test The results show that Bhor OBPm2 selectively chooses chain-like ligands based on the length of carbon chain, and the binding ability of Bhor OBPm2 to ligands is affected by P H. The binding ability of Bhor OBPm2 in neutral environment is obviously stronger than that in acidic environment. Gam OBP1, Cqui OBP1 and Aaeg OBP1 of the cal OBPs family have similar binding cavity structures and ligand selection orientations, but Bhor OBPm2 has larger binding cavity and wider ligand selection due to the absence of a disulfide bond. Therefore, we speculate that Bhor OBPm2 may be an intermediate structure in the evolution of OBPs. Two mutants of Bhor OBPm2, C-ter113 and Y50F, were designed. It was found that the C-terminus of Bhor OBPm2 did not act as a "cap" during the release of the ligand.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S763.38
本文编号：2178511
[Abstract]:Batocera horsfieldi (Hope), also known as Batocera horsfieldi (Hope), is widely distributed in China and is an important stem-borer in forestry. Its sensitive olfactory system plays an important role in host location, mating and oviposition. The odor-binding proteins (OBPs) of insects can carry hydrophobic odor molecules into the atmosphere. Taste receptors play an important role in the specific recognition of informational substances. If the binding and release mechanism of odor-binding proteins and ligand molecules can be clarified, it will provide an important theoretical basis for the study of behavioral regulators. The fluorescence competitive binding characteristics of the main volatiles were studied by computer homology modeling and molecular docking. The structure characteristics of Bhor OBPm2 and its binding and release mechanism with ligand molecules were analyzed by amino acid site-directed mutagenesis technique. The main results were as follows: 1. Cloning of Minus-C OBPs gene, protein expression and purification were successful. The recombinant plasmids P GEX-6P-1-Bhor OBPm1, P ET30a-Bhor OBPm2 and pET17bhor OBPm4.p GEX-6P-1-Bhor OBPm1 were constructed and expressed in the supernatant. The purified protein of Bhor OBPm1 was obtained by GST 4B affinity chromatography. The expression of P ET30a-Bhor OBPm2 and P ET17b-Bhor OBPm4 in inclusion bodies was detected by Ni ion affinity chromatography and DE52 weak anion exchange column, respectively. Bhor OBPm 2 and Bhor OBPm4 were successfully obtained in vitro. 2. The binding properties of three Minus-C OBPs from A. yunnanensis to host volatiles in acidic and neutral environments were determined by using 1-NPN as fluorescent probe. Bhor OBPm1 had weak binding ability to salicylaldehyde in neutral environments, and in acidic and neutral environments. Bhor OBPm2 has strong binding ability with hexanal, trans-2-hexenal and trans-2-hexenol in neutral environment, but weak binding ability with trans-2-hexenol, hexanal and 2-methylbutyraldehyde in acidic environment; Bhor OBPm4 has no binding ability with all volatiles in neutral environment. The binding ability of Bhor OBPm2 was weaker than that of citrone oxides, salicylaldehyde and cis-3-hexenol in acidic environment. 3. Homologous modeling and molecular docking of Bhor OBPm2 were performed using homologous protein Agam OBP1 (2ERB) as template, and the three-dimensional structure of Bhor OBPm2 was modeled. It has six alpha helixes and two disulfide bonds, belonging to Minus-C OBPs. Similar to the C-terminal structure of the template protein Agam OBP1 (2ERB), the amino acid residue Phe123 at the C-terminal of Bhor OBPm2 forms a hydrogen bond with Tyr50 and fixes the C-terminal on the surface of the binding cavity. The docking results of Bhor OBPm2 with 56 odor compounds show that six alpha-helixes form a hydrophobic binding cavity in the center of Bhor OBPm2 and are in the binding cavity. The surface of the binding cavity is mostly hydrophobic, and the small ligand molecules are mainly fixed in the cavity by hydrophobic force. The C-terminal amino acid residues Ile121 and Phe123 form the unique polar region on the surface of the binding cavity. 4. Site-directed mutagenesis of amino acids in Bhor OBPm2 and binding verification fluorescence competitive binding test The results show that Bhor OBPm2 selectively chooses chain-like ligands based on the length of carbon chain, and the binding ability of Bhor OBPm2 to ligands is affected by P H. The binding ability of Bhor OBPm2 in neutral environment is obviously stronger than that in acidic environment. Gam OBP1, Cqui OBP1 and Aaeg OBP1 of the cal OBPs family have similar binding cavity structures and ligand selection orientations, but Bhor OBPm2 has larger binding cavity and wider ligand selection due to the absence of a disulfide bond. Therefore, we speculate that Bhor OBPm2 may be an intermediate structure in the evolution of OBPs. Two mutants of Bhor OBPm2, C-ter113 and Y50F, were designed. It was found that the C-terminus of Bhor OBPm2 did not act as a "cap" during the release of the ligand.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S763.38
【参考文献】
相关期刊论文 前10条
1 刘凤华;王新亮;刘涛;王海玲;;云斑天牛的危害及防治技术[J];安徽农业科学;2014年23期
2 李建庆;杨忠岐;梅增霞;冯斌;王平;王小艺;;释放花绒寄甲对核桃云斑天牛的防治效果[J];中国生物防治学报;2013年02期
3 杨桦;杨伟;杨春平;杨茂发;朱天辉;黄琼;潘武全;;学习经历对川硬皮肿腿蜂寄生云斑天牛幼虫的影响[J];林业科学;2011年08期
4 陈红梅;周俊祥;;基于同源建模的蛋白质结构预测方法的研究[J];河南科学;2009年09期
5 李建庆;杨忠岐;张雅林;梅增霞;张玉荣;王小艺;;利用花绒寄甲防治杨树云斑天牛的研究[J];林业科学;2009年09期
6 李建庆;肖兴翠;李春庆;梅增霞;;云斑天牛对杨树、核桃、白蜡的危害及防治对策[J];滨州学院学报;2008年03期
7 李欣欣;徐晓冬;丹媛媛;张密林;;蛋白质结晶的研究进展[J];生物技术通报;2007年06期
8 喻锦秀;张玉荣;钟武洪;;云斑天牛在杨树上的发生及无公害防治[J];湖南林业科技;2007年05期
9 嵇保中,魏勇,黄振裕;天牛成虫行为研究的现状与展望[J];南京林业大学学报(自然科学版);2002年02期
10 梅爱华,陈京元,吴高云,杜夕森,罗福世;江汉平原杨树害虫种类调查、发生原因及主要害虫防治对策[J];森林病虫通讯;1998年02期
相关硕士学位论文 前1条
1 诸葛飘飘;杨树云斑天牛成虫寄主定位中的信息化学物质[D];华中农业大学;2009年
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