二斑叶螨成虫滞育相关的转录组学和蛋白质组学研究

发布时间：2018-05-08 08:35

本文选题：滞育 + 二斑叶螨　；参考：《南京农业大学》2016年博士论文

【摘要】：温带地区环境的季节性变化使生活在此的节肢动物的发育和生存面临许多挑战。作为变温动物,它们不得不将每年繁殖和生长的时间压缩至温暖季节,并进化出许多可应对极端环境条件的特性,滞育就是其中的一种。滞育是许多节肢动物适应环境变化从而进化出的一种受内源性和中枢神经系统调控的特殊发育阶段,表现为个体中止原有形态发生发育过程。尽管大多数节肢动物的滞育发生在某一特定发育阶段,但不同物种间存在着多样化的滞育策略,包括卵滞育、幼虫滞育、蛹滞育和成虫滞育。滞育过程涉及对个体分子水平、生理水平和形态水平等多方面的调控。明确不同滞育策略间保守和特有的分子水平调控机制是当下滞育研究的热点问题。然而,滞育物种和滞育策略的多样性大大增加了解决这一问题的难度,需要我们对更多具有不同滞育策略的物种开展分子水平的滞育研究。二斑叶螨Tetranychus urticae以滞育受精雌成螨越冬,长期以来其滞育研究一直备受蜱螨学家关注。这不仅由于二斑叶螨滞育存在明显的体色变化,更重要的是其作为一种世界性分布的农业害螨,可危害多达1,100多种植物,因此,明确其滞育机理对于越冬害螨防控手段的研发和应用意义重大。虽然二斑叶螨的滞育研究在形态学、生理学和行为学方面有不少进展,但分子水平的研究进展缓慢。2011年,二斑叶螨的全基因组发表,使其成为第一个完成基因组测序的螯肢动物。基因组测序的完成为二斑叶螨滞育的分子机制研究创造了前所未有的条件。为更系统全面地探究二斑叶螨滞育早期的分子调控机制,本研究以开始排卵的3日龄非滞育型雌成螨(R3)为对照组,选取体色开始变化的3日龄(D3)和开始进入不吃不动阶段的13日龄(D13)两组滞育型雌成螨为处理组,利用RNA-Seq和iTRAQ技术对滞育型雌成螨和非滞育型雌成螨进行了转录组和蛋白质组的比较分析。1.二斑叶螨成虫滞育相关的转录组学研究转录组的分析结果显示3日龄滞育型与非滞育型雌成螨之间有2,777个差异基因,其中滞育型中上调基因1,629个,下调基因1148个;13日龄滞育型与3日龄非滞育型雌成螨之间有差异基因3,277个,其中滞育型中上调基因1,559个,下调基因1678个。对差异基因进行GO, pathway以及Pfam结构域三种功能富集分析发现:1)碳水化合物代谢相关条目和通路在两个比较组中均显著富集,且在D3/R3中上调富集,在D13/R3中下调富集。半胱氨酸肽酶相关条目在D13/R3中下调富集,参与食物消化的半胱氨酸肽酶C1A家族基因结构域在D13/R3中显著富集。我们推测3日龄滞育叶螨的取食和消化活动并未减弱,并将食物吸收转化为碳水化合物储存,以用于滞育期的能量消耗,而在13日龄时这些活动已基本结束。2)解毒代谢相关基因家族细胞色素P450单氧酶、羧基/胆碱酯酶、谷胱甘肽转移酶以及脂质运载蛋白所属的条目、通路以及结构域在两个比较组中均显著富集。表明这几类基因在滞育调控中均发挥重要作用,可能参与滞育早期取食行为的调控。转录组部分的研究表明对于杂食性害虫二斑叶螨,取食和消化行为的调控是滞育早期调控的重要组成部分。2.二斑叶螨成虫滞育相关的蛋白质组学研究作为二斑叶螨的首个定量蛋白质组学研究,我们从3种处理的二斑叶螨总蛋白中共成功鉴定到2,697个蛋白,其中3日龄滞育型与非滞育型雌成螨之间有440个差异蛋白,滞育型中上调244个,下调196个;13日龄滞育型与3日龄非滞育型雌成螨之间有差异蛋白634个,滞育型中上调301个,下调333个。对差异蛋白进行GO富集分析发现核糖体和翻译相关条目在两个比较组中均显著富集且均下调富集,pathway分析发现核糖体通路在第一个比较组中显著富集。这表明蛋白合成过程在雌成螨滞育早期受到抑制。作为细胞中最主要的能量消耗过程,蛋白合成的减弱可以节省大量能量用于其他滞育调控的耗能过程和维持生命的基本能量消耗。另外,结合多项已有研究,我们推测核糖体蛋白S3a在成虫滞育调控中具有非常保守的功能。3.二斑叶螨成虫滞育相关的转录组与蛋白质组联合分析为了从转录和蛋白两个表达水平探究二斑叶螨成虫滞育的分子调控机制,我们将滞育型和非滞育型二斑叶螨的转录组和蛋白质组数据进行关联分析。结果显示:1)转录与蛋白水平的基因表达变化显著相关;2) KOG条目翻译和核糖体的结构及生物合成在蛋白水平富集结果为over且显著下调富集,而在转录水平富集结果为under,表明核糖体和翻译相关基因主要在蛋白水平参与二斑叶螨滞育调控;3) KOG条目能量产生与转化在两个比较组的蛋白水平和D3/R3的转录水平上调富集。表明相较于非滞育叶螨,滞育叶螨在滞育早期通过进行更高强度的有氧氧化合成更多的ATP,以满足其在此阶段的行为活动(取食或寻找越冬场所)和其它生理活动的能量需求;4)KOG条目细胞骨架在两个比较组的转录和蛋白水平均上调富集,对富集到这一条目的基因进行分析发现,肌肉结构、整合素信号通路和微管相关基因在滞育的两个发育节点上调,表明细胞骨架在二斑叶螨成虫滞育早期可能发生重组。4.二斑叶螨成虫滞育相关的神经递质、神经肽和Ca~(2+)信号相关基因分析通过转录组和蛋白质组的GO、pathway以及KOG分析,我们发现不同的分析方法和表达水平均富集到了信号传导相关的条目和通路。因此我们选择与信号传导密切相关的神经递质、神经肽和Ca~(2+)信号进行深入探究。1)谷氨酸为节肢动物重要的神经递质,已有研究发现谷氨酸与二斑叶螨的成虫滞育密切相关。谷氨酸是通过结合谷氨酸受体来参与神经调控,我们从二斑叶螨基因组中鉴定到14个离子型谷氨酸受体(iGluRs) (4 个属于 NMDAR, 3 个属于 AMPAR, 7 个属于 KainateR),其中 6 个 iGluRs在D3/R3中上调。此外代谢型谷氨酸受体(mGluRs)、谷氨酰胺合成酶和谷氨酰胺酶等谷氨酸神经传递相关基因也在D3/R3中上调。因此推测谷氨酸参与调控3日龄滞育雌成螨的突触可塑性。2)对二斑叶螨41个神经肽基因的表达情况进行分析,结果显示转录水平共有16个基因在滞育型与非滞育型二斑叶螨中显著差异表达。我们推测其中3个具有调控取食和消化功能的神经肽proctolin、short neuropeptide F和orcokinin参与滞育型雌成螨滞育早期的取食行为调控。3)虽然Ca~(2+)信号是调控生命活动的“多面手”,但只有少量研究发现Ca~(2+)信号相关基因参与滞育调控。本研究在二斑叶螨基因组范围内鉴定了 Ca~(2+)信号中最重要的两部分-Ca~(2+)结合蛋白和Ca~(2+)转运体基因,然后结合组学数据,发现66个Ca~(2+)结合蛋白和24个Ca~(2+)转运体参与滞育调控。因此推测Ca~(2+)信号在二斑叶螨成虫滞育调控中发挥至关重要的作用。5.类胡萝卜素合成基因在不同色型叶螨中的差异表达二斑叶螨基因组中来自真菌水平转移的6个类胡萝卜素合成基因能否在叶螨中调控类胡萝卜素的合成从而影响体色变化尚无直接证据。具有相同的遗传背景的体色分别为橙色和绿色的的滞育和非滞育型二斑叶螨雌成螨,以及遗传背景相似的体色分别为红色和绿色的非滞育型朱砂叶螨和非滞育型二斑叶螨雌成螨,成为解决以上问题的最佳研究模型。我们首先根据上述的滞育相关组学数据,研究类胡萝卜素合成基因在第一个模型中的差异表达;然后又利用双向电泳技术开展了对第二个模型的蛋白质组比较分析研究,结果显示,与非滞育型二斑叶螨相比较,在滞育型二斑叶螨的两个所选滞育节点,类胡萝卜素合成基因tetur01g11260和tetur01g11270在转录和蛋白水平均显著上调,在朱砂叶螨中tetur01g11260在蛋白水平显著上调。因此,我们认为tetur01g11260和tetur01g11270参与二斑叶螨滞育调控,并可能通过调控类胡萝卜素合成控制滞育型二斑叶螨、非滞育型朱砂叶螨和二斑叶螨三者的体色。
[Abstract]:Seasonal changes in the environment in temperate regions face many challenges to the development and survival of the arthropods living here. As warming animals, they have to compress the time of reproduction and growth every year to warm seasons and evolve many characteristics that can cope with extreme environmental conditions. Diapause is one of the kinds of arthropod movement. A special developmental stage, regulated by the changes of the environment, which is regulated by the endogenous and central nervous system, showing the individual discontinuation of the original development process. Although most arthropod diapause occurs at a particular developmental stage, different species exist in a variety of diapause strategies, including diapause, and young adults. Insect diapause, pupal diapause and adult diapause. Diapause involves the regulation of individual molecular level, physiological level and morphological level. Clear and specific molecular level regulation mechanism between different diapause strategies is a hot issue in diapause research. However, the diversity of diapause species and diapause strategies has been greatly increased. The difficulty of this problem requires us to carry out molecular level diapause studies on more species with different diapause strategies. Two Tetranychus urticae is overwintered by diapause and fertilized female mites. For a long time, the diapause research has been concerned by tick mite experts. This is not only due to the apparent changes in body color, but also in the diapause of the two leaf mite. As a worldwide distribution of agricultural pest mites, it can harm as many as 1100 plants. Therefore, it is important to clarify the mechanism of diapause for the development and application of the means of preventing and controlling the overwintering mite. Although the diapause study of the two leaf mite has a lot of progress in the aspects of morphology, physiology and behavior, the research progress of molecular level is slow. The whole genome of the two leaf mite was published in slow.2011, making it the first sequenced sequenced animal. The completion of genome sequencing has created unprecedented conditions for the molecular mechanism of diapause for the two leaf mite. In order to more systematically and comprehensively explore the molecular regulation mechanism of diapause early for the two leaf mite, this study began to ovulation. The 3 day age non diapause female adult mites (R3) were the control group. The 3 day age (D3) and the 13 day old age (D13) two group of diapause female adult mites were selected as the treatment group. The RNA-Seq and iTRAQ techniques were used to compare the diapause female adult mites and non diapause female adult mites in the transcriptional group and the protein group,.1. two The transcriptional group analysis of the diapause related to the adult diapause showed that there were 2777 different genes between 3 diapause and non diapause female adult mites, of which 1629 were up-regulated and 1148 down regulated in diapause, and 3277 diapause and 3 diapause female adult mites were diapause, of which diapause was diapause. 1559 genes were up-regulated in the type and 1678 down regulated genes. GO, pathway and Pfam domains of three functional enrichment analysis of the differential genes were found: 1) carbohydrate metabolism related items and pathways were significantly enriched in two comparison groups, and were enriched in D3/R3 and decreased in D13/ R3. The related entries of cysteine peptidase were in D13/ The gene domain of the cysteinase C1A family gene in the food digestion was significantly enriched in the R3. We speculated that the feeding and digestion of the 3 day diapause leaf mites did not weaken, and the food absorption was converted into carbohydrate storage for the consumption of diapause, while these activities were basically concluded at the age of 13. .2) the entry of cytochrome P450 monooxygenase, carboxyl / cholinesterase, glutathione transferase, and lipid carrying protein in the detoxification related gene family were significantly enriched in two comparison groups. It showed that these genes played important roles in diapause regulation and may be involved in the early feeding behavior of diapause. The study of the transcriptome part showed that the control of the two leaf mite, feeding and digestion of the omnivorous insect, the important component of the early regulation of diapause, the.2. two leaf mite diapause related proteomics as the first quantitative proteomics study of the two leaf mite, we from 3 treatments of the total eggs of two leaf mite. 2697 proteins were successfully identified in the Chinese Communist Party, of which there were 440 differential proteins between 3 diapause and non diapause female adult mites, 244 diapause types, 196 down-regulation, 634 diapause and 3 day old non diapause female adult mites, 301 and 333 diapause types. The difference protein was enriched by GO enrichment. It was found that the ribosome and translation related items were enriched in two comparison groups and were all down-regulated. Pathway analysis found that the ribosome pathway was significantly enriched in the first comparison group. This indicates that the protein synthesis process is inhibited in the early stage of the diapause of female adult mites. As the most important process of energy consumption in the cells, the decrease of protein synthesis can be reduced. In order to save a lot of energy for the energy consumption process of other diapause regulation and to maintain the basic energy consumption of life, combined with a number of previous studies, we speculate that the ribosomal protein S3a has a very conservative function in the regulation of diapause of the adult.3. two diapause related to the diapause and proteome analysis in order to be transcribed. Two expressions of protein and protein were used to investigate the molecular regulation mechanism of diapause in adult two leaf mite. We correlated the transcriptional and proteomic data of diapause and non diapause type two leaf mite. The results showed that: 1) transcription was significantly related to gene expression changes at protein level; 2) KOG entry translation and ribosome structure and biosynthesis. The result of protein level enrichment is over and significantly down-regulated and enriched at the transcriptional level of under, indicating that ribosome and translation related genes are mainly involved in the regulation of diapause of two leaf mite at the protein level; 3) the energy generation and transformation of KOG entries are up-regulated in the level of protein and the transcriptional level of D3/R3 in two comparative groups. Compared with non diapause leaf mite, diapause mite can synthesize more ATP by aerobic oxidation at the early stage of diapause, to meet its activity activity at this stage (eating or looking for overwintering sites) and energy requirements for other physiological activities; 4) the KOG entry cytoskeleton is enriched in the transcriptional and protein levels of the two comparative groups and is rich in the rich. Analysis of this target gene found that muscle structure, integrin signaling pathway and microtubule related genes were up-regulated in two diapause development nodes, indicating that the cytoskeleton may occur in the early stage of the diapause of the two reconstituted.4. two leaf mite. The neuropeptide and Ca~ (2+) signal related gene analysis Through the analysis of GO, pathway, and KOG in the transcriptional and proteome groups, we found that different analytical methods and levels of expression are enriched in signal transduction related items and pathways. Therefore, we choose neurotransmitters, neuropeptides and Ca~ (2+) signals, which are closely related to signal transduction, to explore.1 in depth.) glutamic acid is important for arthropods. Neurotransmitter, it has been found that glutamate is closely related to the adult diapause of two Tetranychus. Glutamic acid is involved in neural regulation by binding glutamate receptors. We identified 14 ionic glutamate receptors (iGluRs) (4 NMDAR, 3 AMPAR, 7 KainateR) from the genome of the mite (6 I). GluRs is up-regulated in D3/R3. In addition, the glutamic acid receptor (mGluRs), glutamine synthetase and glutaminase are also up regulated in D3/R3. Therefore, it is speculated that glutamic acid is involved in the regulation of the synaptic plasticity of the 3 day diapause female adult mites, and the expression of 41 neuropeptide genes in the two leaf mite is analyzed. The results showed that 16 genes were significantly different in diapause and non diapause two mite. We speculated that 3 of them had neuropeptide proctolin regulating feeding and digestive functions, short neuropeptide F and orcokinin participated in the regulation of.3 for the feeding behavior of diapause female adult mites at the early stage of diapause, although the Ca~ (2+) signal was regulated by the Ca~ (2+) signal. But only a few studies have found that Ca~ (2+) signal related genes are involved in diapause regulation. This study identified the most important two parts of the Ca~ (2+) signal, -Ca~ (2+) binding protein and Ca~ (2+) transporter gene in the Ca~ (2+) signal genome, and then found 66 Ca~ (2+) binding protein and 24 in combination with the data of the group. Ca~ (2+) transporter participates in diapause regulation. Therefore, it is speculated that Ca~ (2+) signal plays an important role in the regulation of diapause of two leaf mite adult. The difference expression of.5. carotenoid synthetic gene in different color mites is expressed in the genome of 6 carotenoids from the level of fungus level transfer in the genome of two leaf mite. There is no direct evidence for the synthesis of the controlled carotenoids and the changes in the body color. The color of the same genetic background is the orange and green diapause and the non diapause, the female mites of the two leaf mite, and the similar color of the genetic background are the red and green non diapause and the non diapause female two mite. To become the best research model to solve the above problems, we first study the differential expression of the carotenoid synthesis gene in the first model according to the diapause related data, and then use two dimensional electrophoresis to carry out the analysis of the protein group ratio of the second models. The results show that the two spots of the non diapause type are compared with that of the non diapause. Ye Manxiang compared the two diapause node of diapause type two leaf mite, the carotenoid synthesis gene tetur01g11260 and tetur01g11270 increased significantly in both the transcription and protein levels, and the protein level of tetur01g11260 was significantly up-regulated in the acarus cinnabara. Therefore, we think that tetur01g11260 and tetur01g11270 are involved in the diapause of two leaf mite. The body color of diapause type two spider mite, non diapause type spider mite and two spotted spider mites can be controlled by regulating the synthesis of carotenoids.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433

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