暗黑鳃金龟雌雄触角差异蛋白质组学分析

发布时间：2018-11-17 07:14

【摘要】：暗黑鳃金龟（Holotrichia parallela Motschulsky）属于鞘翅目鳃金龟科，其幼虫称蛴螬，是重要的地下害虫，对农作物、蔬菜和果树能引发严重的为害。因此，有效的防治暗黑鳃金龟已成为亟待解决的问题。嗅觉系统对于昆虫寻找寄主、配偶等行为至关重要，了解昆虫嗅觉系统将有助于研发出新的防治策略来治理害虫，包括利用昆虫的嗅觉识别机制驱散或吸引害虫以达到防治的目的。差异蛋白质组学能够揭示嗅觉相关蛋白，是解析触角嗅觉分子识别机制的重要措施。为了明确暗黑鳃金龟的触角嗅觉识别机制，我们应用蛋白质双向电泳(2-DE)、质谱鉴定以及生物信息学分析技术对暗黑鳃金龟雌雄触角蛋白进行差异分析和功能验证。蛋白样品的制备是2-DE蛋白质组学研究的关键环节之一。为了建立一个稳定的触角蛋白质组学研究平台，我们对四种触角蛋白的制备方法（TCA/丙酮沉淀法、饱和酚法、PEG法和直接提取法）进行了分析和优化。根据蛋白产率、SDS-PAGE电泳以及蛋白点数的分析发现，TCA/丙酮沉淀法是提取触角蛋白的最优方法。应用软件ImageMaster2D Platinum7.0对银染后的蛋白质双向电泳图谱进行分析后发现，每张凝胶上能检测到约1100个蛋白点。差异蛋白分析表明，雌雄触角里总共有47个蛋白点发生了显著性（1.5fold，p-value 0.05）变化。其中，25个蛋白点在雄虫触角里上调表达，22个蛋白点在雌虫触角里上调表达。为了鉴定触角里的差异表达蛋白，运用MALDI-TOF/TOF MS质谱技术对从凝胶上分离下来的蛋白点进行分析。47个差异表达蛋白中有35个被成功鉴定。在这些蛋白中，65.7%的蛋白与碳水化合物和能量代谢、抗氧化系统、转运以及氨基酸和核苷酸代谢有关。部分嗅觉相关蛋白首次被鉴定，包括肽聚糖识别蛋白-1（spot16），酰基辅酶A脱氢酶(spot26)，磷酸丙酮异构酶类似物(spot29)，醛酮还原酶类似物(spot34)、丝氨酸蛋白酶(spot21)，气味结合蛋白2(spot38)，，乙醛脱氢酶(spot14)，细胞色素P450（spot12）等。依据KEGG和Gene Ontology数据库和软件对差异表达蛋白进行生物信息学分析，将鉴定出的蛋白共分成8类。其中，20.0%的蛋白与碳水化合物和能量代谢相关，17.1%的蛋白与抗氧化系统相关，14.3%的蛋白与转运相关,14.3%的蛋白与氨基酸和核苷酸代谢相关。另外还包括11.4%的细胞骨架蛋白和8.6%与蛋白质折叠相关的蛋白。对鉴定的6个嗅觉相关蛋白进行了蛋白质互作网络分析，包括热激蛋白70同源物-3、丝氨酸蛋白酶和醛酮还原酶类似物，以及触角里含量丰富的细胞色素P450、乙醛脱氢酶和谷氨酰胺合成酶，为蛋白质功能的进一步研究创造了条件。应用实时荧光定量PCR技术检测了6个嗅觉相关蛋白在转录水平上的变化，结果显示mRNA与蛋白质水平的变化基本一致。
[Abstract]:(Holotrichia parallela Motschulsky) belongs to the family Coleoptera, and its larvae are called grub, which is an important underground pest, which can cause serious damage to crops, vegetables and fruit trees. Therefore, the effective prevention and cure dark gills golden tortoise has become the question which urgently needs to be solved. The olfactory system is essential for insects to find hosts, mates and other behaviors. Understanding the insect olfactory system will help to develop new control strategies to control pests. Including the use of insect olfactory recognition mechanism to disperse or attract pests for control purposes. Differential proteomics, which can reveal olfactory related proteins, is an important measure to elucidate the molecular recognition mechanism of antennae olfaction. In order to clarify the mechanism of olfactory recognition of the antennae, we used protein two-dimensional electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics analysis techniques to analyze and verify the function of male and female antennae proteins. The preparation of protein sample is one of the key links in 2-DE proteomics. In order to establish a stable platform for the study of antennal proteomics, four methods for the preparation of antennal proteins (TCA/ acetone precipitation method, saturated phenol method, PEG method and direct extraction method) were analyzed and optimized. According to protein yield, SDS-PAGE electrophoresis and protein number analysis, it was found that TCA/ acetone precipitation method was the best method for extracting antennal proteins. Using software ImageMaster2D Platinum7.0 to analyze the two dimensional electrophoresis patterns of silver-stained proteins, it was found that about 1,100 protein spots could be detected on each gel. The results of differential protein analysis showed that there were significant changes in 47 protein spots (1.5 foldsp-value 0.05) in the male and female antennae. Among them, 25 protein spots were up-regulated in male antennae and 22 protein spots were up-regulated in female antennae. In order to identify differentially expressed proteins in the antennae, the protein spots isolated from the gel were analyzed by MALDI-TOF/TOF MS mass spectrometry. 35 of the 47 differentially expressed proteins were successfully identified. Of these proteins, 65.7% were related to carbohydrate and energy metabolism, antioxidant system, transport and metabolism of amino acids and nucleotides. Some olfactory related proteins have been identified for the first time, including peptidoglycan recognition protein-1 (spot16), acyl-coenzyme A dehydrogenase (spot26), phosphoacetone isomerase analogues (spot29), aldehyde-ketone reductase analogues (spot34), serine protease (spot21), etc. Smell binding protein 2 (spot38), acetaldehyde dehydrogenase (spot14), cytochrome P450 (spot12), etc. According to KEGG and Gene Ontology database and software, the differentially expressed proteins were analyzed by bioinformatics, and the identified proteins were classified into 8 categories. Among them, 20.0% of proteins were related to carbohydrate and energy metabolism, 17.1% to antioxidant system, 14.3% to transport, 14.3% to amino acid and nucleotide metabolism. It also included 11.4% cytoskeleton protein and 8.6% protein related to protein folding. Six olfactory related proteins were identified by protein interaction network analysis, including heat shock protein 70 homologues-3, serine protease and aldehyde-ketone reductase analogues, and cytochrome P450, which is abundant in the antennae. Aldehyde dehydrogenase and glutamine synthase create conditions for further study of protein function. The changes of six olfactory related proteins at the transcriptional level were detected by real-time fluorescence quantitative PCR. The results showed that the changes of mRNA and protein levels were basically the same.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.5

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