姜黄素胁迫下朱砂叶螨mRNA测序及响应基因分析

发布时间：2018-01-19 01:14

本文关键词： 姜黄素朱砂叶螨数字基因表达谱钙调蛋白磷脂酶A2　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：本实验室通过筛选一系列化合物发现,源于中草药植物姜黄的活性物质姜黄素对朱砂叶螨表现出良好的杀螨活性,具有开发成绿色杀螨剂的潜力。因此明确姜黄素杀螨的分子机制,从而以姜黄素的作用靶标为依据,以姜黄素为模板,进行分子优化,将有助于新的绿色杀螨剂的研制。目前虽然在姜黄素的杀螨机理方面取得了一定的进展,但是杀螨的分子机制仍不清楚。并且,朱砂叶螨基因信息以及姜黄素杀螨机制密切相关的应答基因的缺乏一直是该领域的一大阻碍。本研究采用了数字基因表达谱升级版测序技术对朱砂叶螨响应姜黄素胁迫的分子机制以及响应基因进行了研究,扩展了参与姜黄素杀螨的基因信息资源,为进一步全面阐述姜黄素的杀螨机理提供了思路,同时丰富了天然活性物质杀虫/螨机制的研究内容。主要的研究成果如下:1.明确了姜黄素对朱砂叶螨的LC50为2.642mg/m L,并表现出类似神经毒剂的中毒症状姜黄素的生物活性测定结果表明,姜黄素对朱砂叶螨48 h的毒力回归方程为y=-0.278+0.659x,致死中浓度LC50为2.642 mg/m L,95%的置信区间为1.680~6.082。玻片浸渍法和叶碟喷雾法处理朱砂叶螨后进行症状观察发现,朱砂叶螨的中毒症状大致可分为:静止期(0 h-4h);复苏兴奋期(4 h-48 h);麻痹昏迷期(48 h-72 h);死亡期(72 h-144 h)。这跟神经毒剂的致毒症状有一定的相似性。并根据中毒症状明确了姜黄素作用的重要时间节点24 h和48 h,为下步测序实验提供理论依据。2.获得并验证了数字基因表达谱测序结果,明确了姜黄素处理下朱砂叶螨响应基因参与的主要生物学过程处理组和溶剂对照组测序分别得到53 419 438条和52 286 859条Reads。24 h和48 h的差异表达基因分别为15450条和15371条。满足偏离概率值≥0.8并且log2Ratio≥1的显著差异基因分别有111条和96条。此外,荧光定量证实了m RNA测序结果的准确性,同时说明参与验证的15条基因也确实对姜黄素发生了响应。GO功能显著性富集分析明确了显著差异基因参与的主要生物学过程包括生理过程、代谢过程、单生物过程、细胞凋亡、连接和催化、离子跨膜转运、神经传导等。KEGG通路富集分析罗列了富集程度排名前20的生化代谢途径和信号转导途径,其中内质网蛋白质加工和MAPK信号转导通路是24 h和48 h均具有的最具代表性的生化途径。另外与化合物分解代谢、信号传导、细胞吞噬等作用有关的基因约占整个KEGG通路基因的64%。这些GO条目和KEGG条目里所包含的基因很可能与姜黄素杀螨机制密切相关。3.获得23条与螨体代谢、信号传导等相关的显著差异基因,测定了谷胱甘肽-S-转移酶活性,明确了螨体解毒代谢情况通过RPKM和Noiseq算法,筛选出两处理时间段的显著差异基因总条数为190条,共有基因为17条。获得了医学上姜黄素的靶标丝氨酸/苏氨酸激酶以及与姜黄素的医学靶标相关的鸟苷酸激酶、硫氧还蛋白样4A和Ras GTP酶。另外本研究还获得了很多与钙离子相关的酶,如磷脂酶C家族、磷脂酶A2、钙调蛋白、钙联接蛋白和内质网应激相关蛋白1,其中磷脂酶A2和钙调蛋白是重要的信号传导类蛋白也是差异表达最明显的基因,可以作为姜黄素杀螨作用的候选靶标。同时获得了GSTs基因,明确了谷胱甘肽-S-转移酶随姜黄素处理时间的变化情况,初步说明朱砂叶螨体内姜黄素的减少并非是与谷胱甘肽硫转移酶发生作用所致。4.克隆并分析了朱砂叶螨钙调蛋白(calmodulin,Tc Ca M)和磷脂酶A2(phospholipase A2,Tc PLA2)基因的c DNA序列克隆获得了Tc Ca M和Tc PLA2基因c DNA序列,分别命名为Tc Ca M、Tc PLA2,登录号分别为:KY436226、KY436225。预测蛋白质相对分子量分别为19.03KDa和12.04KDa,等电点分别为4.32和4.97。另外通过信号肽、蛋白跨膜结构、糖基化位点和结构域预测发现,钙调蛋白和磷脂酶A2均无信号肽和跨膜结构,均为非跨膜蛋白。钙调蛋白具有四个EF-hand结构域和两个糖基化位点,磷脂酶A2只有一个糖基化位点,未发现结构域。进化树结果显示这两条基因所处的分支分别与二斑叶螨最接近,该结果与传统意义上的分类结果一致。5.朱砂叶螨Tc Ca M和Tc PLA2基因在不同螨态均有表达,对姜黄素杀螨机制的研究具有重要价值Tc Ca M和Tc PLA2基因在朱砂叶螨不同螨态的表达表明:这两个基因在朱砂叶螨的四个螨态中均有表达,并且幼螨、若螨期的表达量要显著高于卵期和成螨期。钙调蛋白基因的表达由高到低依次为若螨幼螨成螨卵,除幼螨、若螨差异不显著外,其余均呈显著性差异。磷脂酶A2基因的表达量高低依次为幼螨若螨成螨卵,各螨态均成显著性差异。Tc Ca M和Tc PLA2基因因其特殊的功能和在朱砂叶螨不同发育阶段的广泛存在使之成为维持螨体正常生命活动的重要蛋白,因此它们的抑制和活化都将对螨体产生显著影响,钙调蛋白和磷脂酶A2在姜黄素的杀螨机制中具有很重要的研究价值。
[Abstract]:The laboratory by screening a series of compounds that originated from the herbal plant turmeric active substances of curcumin on Zhu Shaye mites showed good acaricidal activity, has developed into a green acaricide. The molecular mechanism of curcumin so clear potential acaricide targets, thus curcumin as the basis, with curcumin as a template for the molecular optimization, will help to develop new green acaricides. Although the acaricidal mechanism of curcumin has made some progress, but the molecular mechanism of acaricidal remains unclear. Moreover, the lack of response gene tetranychuscinnabarinus gene information and closely related to the killing mechanism of curcumin has been a major obstacles in this field. This study adopts digital gene expression profile sequencing technology upgrades to tetranychuscinnabarinus molecular response mechanism of curcumin and stress response genes were studied and extended In the genetic information resources curcumin acaricide, provides ideas for further comprehensive exposition of acaricidal mechanism of curcumin, and enrich the research content of natural insecticidal / mite mechanism. The main results are as follows: 1. the LC50 of curcumin to tetranychuscinnabarinus is 2.642mg/m L, and showed similar results for determination of nerve agent the symptoms of poisoning the bioactivities of curcumin showed that curcumin on toxicity regression equation of 48 h tetranychuscinnabarinus y=-0.278+0.659x, lethal concentration LC50 was 2.642 mg/m L, 95% confidence interval 1.680~6.082. slide impregnation method and leaf disc tetranychuscinnabarinus treatment spray method after symptoms were observed, symptoms of poisoning tetranychuscinnabarinus roughly can be divided into: stationary phase (0 h-4h); recovery of exciting period (4 h-48 h); paralysis coma period (48 h-72 h); death period (72 h-144 h). This is similar to the nerve agent poisoning symptoms According to the symptoms of poisoning. The important time node curcumin 24 h and 48 h, for the next step sequencing experiments provide a theoretical basis for.2. to obtain and verify the digital gene expression profile sequencing results, the response of genes involved in the biological process of the main treatment group and solvent control group were respectively 53419438 and 52286859 Reads.24 h h and 48 differentially expressed genes were 15450 and 15371, tetranychuscinnabarinus curcumin treatment. Meet the deviation probability value is more than 0.8 and 1 log2Ratio genes were respectively 111 and 96. In addition, fluorescence quantitative m confirmed the accuracy of the results of RNA sequencing, and 15 genes involved in verification does the curcumin occurred in response to the.GO function significant enrichment analysis clearly the main differences of genes involved in the biological processes including physiological process, metabolic process, biological single Process, apoptosis, connection and catalysis, ion transport across the membrane, nerve conduction and.KEGG pathway enrichment analysis lists the enrichment degree of the top 20 biochemical metabolic pathways and signal transduction pathways, including endoplasmic reticulum protein processing and signal transduction pathway of MAPK is 24 h and 48 h are some of the most representative biochemical pathways. In addition to the compound decomposition metabolism, signal transduction, cell phagocytosis gene effects about which contains the entire KEGG pathway genes 64%. these GO and KEGG entries in the gene may kill mite mechanism and curcumin is closely related to.3. was 23 and the mites metabolism genes were related to signal transduction, glutathione -S- transferase activity determination, clear the mites detoxification through the RPKM and Noiseq algorithms, selected genes differ significantly between the two treatment period the total number of 190, a total of 17 genes. The target serine / threonine kinase and medicine curcumin and curcumin medical target associated guanylate kinase, thioredoxin like 4A and Ras GTP enzyme. In addition this study also received a lot of calcium ions and related enzymes, such as phospholipase C family, phospholipase A2, calmodulin, calmegin and the endoplasmic reticulum stress related protein 1, including phospholipase A2 and calmodulin gene is the most obvious signal transduction proteins is an important differential expression, can be used as a candidate target of curcumin and miticides. At the same time GSTs gene has been obtained, the changes of glutathione -S- transferase with curcumin treatment time, preliminary description of cinnabar spider mite in curcumin reduction is not analyzed tetranychuscinnabarinus and Calmodulin effect caused by.4. cloning and glutathione-s transferase (calmodulin, Tc Ca M (phospholipase) and phospholipase A2 A2, Tc PLA2) C DNA cloned gene sequence was Tc Ca M and Tc PLA2 C gene DNA sequence, named Tc Ca M, Tc PLA2, the accession number was KY436226, the relative molecular weight of KY436225. and 19.03KDa for the prediction of protein 12.04KDa, isoelectric point was 4.32 and 4.97. respectively by signal protein peptide, transmembrane structure, glycosylation sites and domain prediction found that calmodulin and phospholipase A2 have no signal peptide and transmembrane structure are non transmembrane protein. Calmodulin has four EF-hand domains and two glycosylation sites, phospholipase A2 only a glycosylation site not found domain. The phylogenetic tree showed that the branch of the two genes were the most close to the two spotted spider mite, the classification results with the results of the traditional sense of the.5. Tc Ca M tetranychuscinnabarinus and Tc PLA2 gene in different states were mite killing mechanism of curcumin, the research This has important value of Tc M and Ca Tc expression of PLA2 gene in tetranychuscinnabarinus different mite state: the two genes in four states mite Tetranychus cinnabarinus were expressed, and the expression of larval mites, if mites period was significantly higher than the egg and adult period. The expression of calmodulin gene from high to low as if the young adult mites mite eggs, in addition to David mite nymphs, the difference was not significant, the rest showed a significant difference. The expression level of phospholipase A2 gene in young adult mites mites if the mite eggs, there were significant differences in.Tc state of Ca M and Tc PLA2 gene its special function and in tetranychuscinnabarinus different developmental stages exist widely and has become an important protein to maintain the normal life activities of the mites, so their inhibition and activation will have a significant impact on the mites, calmodulin and phospholipase A2 has very important value in the research on price acaricidal mechanism of curcumin.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S482.52

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