發(fā)布時間：2018-05-08 08:35

  本文選題：滯育 + 二斑葉螨�。� 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：溫帶地區(qū)環(huán)境的季節(jié)性變化使生活在此的節(jié)肢動物的發(fā)育和生存面臨許多挑戰(zhàn)。作為變溫動物,它們不得不將每年繁殖和生長的時間壓縮至溫暖季節(jié),并進化出許多可應(yīng)對極端環(huán)境條件的特性,滯育就是其中的一種。滯育是許多節(jié)肢動物適應(yīng)環(huán)境變化從而進化出的一種受內(nèi)源性和中樞神經(jīng)系統(tǒng)調(diào)控的特殊發(fā)育階段,表現(xiàn)為個體中止原有形態(tài)發(fā)生發(fā)育過程。盡管大多數(shù)節(jié)肢動物的滯育發(fā)生在某一特定發(fā)育階段,但不同物種間存在著多樣化的滯育策略,包括卵滯育、幼蟲滯育、蛹滯育和成蟲滯育。滯育過程涉及對個體分子水平、生理水平和形態(tài)水平等多方面的調(diào)控。明確不同滯育策略間保守和特有的分子水平調(diào)控機制是當(dāng)下滯育研究的熱點問題。然而,滯育物種和滯育策略的多樣性大大增加了解決這一問題的難度,需要我們對更多具有不同滯育策略的物種開展分子水平的滯育研究。二斑葉螨Tetranychus urticae以滯育受精雌成螨越冬,長期以來其滯育研究一直備受蜱螨學(xué)家關(guān)注。這不僅由于二斑葉螨滯育存在明顯的體色變化,更重要的是其作為一種世界性分布的農(nóng)業(yè)害螨,可危害多達1,100多種植物,因此,明確其滯育機理對于越冬害螨防控手段的研發(fā)和應(yīng)用意義重大。雖然二斑葉螨的滯育研究在形態(tài)學(xué)、生理學(xué)和行為學(xué)方面有不少進展,但分子水平的研究進展緩慢。2011年,二斑葉螨的全基因組發(fā)表,使其成為第一個完成基因組測序的螯肢動物�；�因組測序的完成為二斑葉螨滯育的分子機制研究創(chuàng)造了前所未有的條件。為更系統(tǒng)全面地探究二斑葉螨滯育早期的分子調(diào)控機制,本研究以開始排卵的3日齡非滯育型雌成螨(R3)為對照組,選取體色開始變化的3日齡(D3)和開始進入不吃不動階段的13日齡(D13)兩組滯育型雌成螨為處理組,利用RNA-Seq和iTRAQ技術(shù)對滯育型雌成螨和非滯育型雌成螨進行了轉(zhuǎn)錄組和蛋白質(zhì)組的比較分析。1.二斑葉螨成蟲滯育相關(guān)的轉(zhuǎn)錄組學(xué)研究轉(zhuǎn)錄組的分析結(jié)果顯示3日齡滯育型與非滯育型雌成螨之間有2,777個差異基因,其中滯育型中上調(diào)基因1,629個,下調(diào)基因1148個;13日齡滯育型與3日齡非滯育型雌成螨之間有差異基因3,277個,其中滯育型中上調(diào)基因1,559個,下調(diào)基因1678個。對差異基因進行GO, pathway以及Pfam結(jié)構(gòu)域三種功能富集分析發(fā)現(xiàn):1)碳水化合物代謝相關(guān)條目和通路在兩個比較組中均顯著富集,且在D3/R3中上調(diào)富集,在D13/R3中下調(diào)富集。半胱氨酸肽酶相關(guān)條目在D13/R3中下調(diào)富集,參與食物消化的半胱氨酸肽酶C1A家族基因結(jié)構(gòu)域在D13/R3中顯著富集。我們推測3日齡滯育葉螨的取食和消化活動并未減弱,并將食物吸收轉(zhuǎn)化為碳水化合物儲存,以用于滯育期的能量消耗,而在13日齡時這些活動已基本結(jié)束。2)解毒代謝相關(guān)基因家族細胞色素P450單氧酶、羧基/膽堿酯酶、谷胱甘肽轉(zhuǎn)移酶以及脂質(zhì)運載蛋白所屬的條目、通路以及結(jié)構(gòu)域在兩個比較組中均顯著富集。表明這幾類基因在滯育調(diào)控中均發(fā)揮重要作用,可能參與滯育早期取食行為的調(diào)控。轉(zhuǎn)錄組部分的研究表明對于雜食性害蟲二斑葉螨,取食和消化行為的調(diào)控是滯育早期調(diào)控的重要組成部分。2.二斑葉螨成蟲滯育相關(guān)的蛋白質(zhì)組學(xué)研究作為二斑葉螨的首個定量蛋白質(zhì)組學(xué)研究,我們從3種處理的二斑葉螨總蛋白中共成功鑒定到2,697個蛋白,其中3日齡滯育型與非滯育型雌成螨之間有440個差異蛋白,滯育型中上調(diào)244個,下調(diào)196個;13日齡滯育型與3日齡非滯育型雌成螨之間有差異蛋白634個,滯育型中上調(diào)301個,下調(diào)333個。對差異蛋白進行GO富集分析發(fā)現(xiàn)核糖體和翻譯相關(guān)條目在兩個比較組中均顯著富集且均下調(diào)富集,pathway分析發(fā)現(xiàn)核糖體通路在第一個比較組中顯著富集。這表明蛋白合成過程在雌成螨滯育早期受到抑制。作為細胞中最主要的能量消耗過程,蛋白合成的減弱可以節(jié)省大量能量用于其他滯育調(diào)控的耗能過程和維持生命的基本能量消耗。另外,結(jié)合多項已有研究,我們推測核糖體蛋白S3a在成蟲滯育調(diào)控中具有非常保守的功能。3.二斑葉螨成蟲滯育相關(guān)的轉(zhuǎn)錄組與蛋白質(zhì)組聯(lián)合分析為了從轉(zhuǎn)錄和蛋白兩個表達水平探究二斑葉螨成蟲滯育的分子調(diào)控機制,我們將滯育型和非滯育型二斑葉螨的轉(zhuǎn)錄組和蛋白質(zhì)組數(shù)據(jù)進行關(guān)聯(lián)分析。結(jié)果顯示:1)轉(zhuǎn)錄與蛋白水平的基因表達變化顯著相關(guān);2) KOG條目翻譯和核糖體的結(jié)構(gòu)及生物合成在蛋白水平富集結(jié)果為over且顯著下調(diào)富集,而在轉(zhuǎn)錄水平富集結(jié)果為under,表明核糖體和翻譯相關(guān)基因主要在蛋白水平參與二斑葉螨滯育調(diào)控;3) KOG條目能量產(chǎn)生與轉(zhuǎn)化在兩個比較組的蛋白水平和D3/R3的轉(zhuǎn)錄水平上調(diào)富集。表明相較于非滯育葉螨,滯育葉螨在滯育早期通過進行更高強度的有氧氧化合成更多的ATP,以滿足其在此階段的行為活動(取食或?qū)ふ以蕉瑘鏊?和其它生理活動的能量需求;4)KOG條目細胞骨架在兩個比較組的轉(zhuǎn)錄和蛋白水平均上調(diào)富集,對富集到這一條目的基因進行分析發(fā)現(xiàn),肌肉結(jié)構(gòu)、整合素信號通路和微管相關(guān)基因在滯育的兩個發(fā)育節(jié)點上調(diào),表明細胞骨架在二斑葉螨成蟲滯育早期可能發(fā)生重組。4.二斑葉螨成蟲滯育相關(guān)的神經(jīng)遞質(zhì)、神經(jīng)肽和Ca~(2+)信號相關(guān)基因分析通過轉(zhuǎn)錄組和蛋白質(zhì)組的GO、pathway以及KOG分析,我們發(fā)現(xiàn)不同的分析方法和表達水平均富集到了信號傳導(dǎo)相關(guān)的條目和通路。因此我們選擇與信號傳導(dǎo)密切相關(guān)的神經(jīng)遞質(zhì)、神經(jīng)肽和Ca~(2+)信號進行深入探究。1)谷氨酸為節(jié)肢動物重要的神經(jīng)遞質(zhì),已有研究發(fā)現(xiàn)谷氨酸與二斑葉螨的成蟲滯育密切相關(guān)。谷氨酸是通過結(jié)合谷氨酸受體來參與神經(jīng)調(diào)控,我們從二斑葉螨基因組中鑒定到14個離子型谷氨酸受體(iGluRs) (4 個屬于 NMDAR, 3 個屬于 AMPAR, 7 個屬于 KainateR),其中 6 個 iGluRs在D3/R3中上調(diào)。此外代謝型谷氨酸受體(mGluRs)、谷氨酰胺合成酶和谷氨酰胺酶等谷氨酸神經(jīng)傳遞相關(guān)基因也在D3/R3中上調(diào)。因此推測谷氨酸參與調(diào)控3日齡滯育雌成螨的突觸可塑性。2)對二斑葉螨41個神經(jīng)肽基因的表達情況進行分析,結(jié)果顯示轉(zhuǎn)錄水平共有16個基因在滯育型與非滯育型二斑葉螨中顯著差異表達。我們推測其中3個具有調(diào)控取食和消化功能的神經(jīng)肽proctolin、short neuropeptide F和orcokinin參與滯育型雌成螨滯育早期的取食行為調(diào)控。3)雖然Ca~(2+)信號是調(diào)控生命活動的“多面手”,但只有少量研究發(fā)現(xiàn)Ca~(2+)信號相關(guān)基因參與滯育調(diào)控。本研究在二斑葉螨基因組范圍內(nèi)鑒定了 Ca~(2+)信號中最重要的兩部分-Ca~(2+)結(jié)合蛋白和Ca~(2+)轉(zhuǎn)運體基因,然后結(jié)合組學(xué)數(shù)據(jù),發(fā)現(xiàn)66個Ca~(2+)結(jié)合蛋白和24個Ca~(2+)轉(zhuǎn)運體參與滯育調(diào)控。因此推測Ca~(2+)信號在二斑葉螨成蟲滯育調(diào)控中發(fā)揮至關(guān)重要的作用。5.類胡蘿卜素合成基因在不同色型葉螨中的差異表達二斑葉螨基因組中來自真菌水平轉(zhuǎn)移的6個類胡蘿卜素合成基因能否在葉螨中調(diào)控類胡蘿卜素的合成從而影響體色變化尚無直接證據(jù)。具有相同的遺傳背景的體色分別為橙色和綠色的的滯育和非滯育型二斑葉螨雌成螨,以及遺傳背景相似的體色分別為紅色和綠色的非滯育型朱砂葉螨和非滯育型二斑葉螨雌成螨,成為解決以上問題的最佳研究模型。我們首先根據(jù)上述的滯育相關(guān)組學(xué)數(shù)據(jù),研究類胡蘿卜素合成基因在第一個模型中的差異表達;然后又利用雙向電泳技術(shù)開展了對第二個模型的蛋白質(zhì)組比較分析研究,結(jié)果顯示,與非滯育型二斑葉螨相比較,在滯育型二斑葉螨的兩個所選滯育節(jié)點,類胡蘿卜素合成基因tetur01g11260和tetur01g11270在轉(zhuǎn)錄和蛋白水平均顯著上調(diào),在朱砂葉螨中tetur01g11260在蛋白水平顯著上調(diào)。因此,我們認為tetur01g11260和tetur01g11270參與二斑葉螨滯育調(diào)控,并可能通過調(diào)控類胡蘿卜素合成控制滯育型二斑葉螨、非滯育型朱砂葉螨和二斑葉螨三者的體色。
[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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S433

【相似文獻】

相關(guān)期刊論文 前10條

1 劉景毅;林健榮;;家蠶滯育及滯育解除機制研究進展[J];廣東蠶業(yè);2011年03期

2 黎萬順;陳斌;何正波;;蔥蠅非滯育、冬滯育和夏滯育蛹發(fā)育和形態(tài)特征比較[J];昆蟲學(xué)報;2012年07期

3 蔣猷龍;;桑蠶卵滯育機理研究進展[J];蠶桑通報;1980年03期

4 吳敦肅 ,王高順;桑蠶卵的滯育[J];廣西蠶業(yè)通訊;1982年02期

5 蘇樹鑫;滕國良;伍錫生;;家蠶越年蠶卵冬季解除滯育的調(diào)查[J];江蘇蠶業(yè);1982年03期

6 島田順;沈衛(wèi)德;;家蠶咽側(cè)體在誘導(dǎo)卵滯育中的作用[J];國外農(nóng)學(xué)-蠶業(yè);1986年04期

7 沈榮武;薛芳森;;二化螟滯育發(fā)生的特點及其與化性的關(guān)系[J];江西植保;1987年02期

8 李亞新,張乃鑫;偽鈍綏螨滯育的研究[J];植物保護;1990年05期

9 Eiichi Kuwano;承憲成;;用幾種咪唑解除天蠶卵的滯育[J];國外農(nóng)學(xué)-蠶業(yè);1992年04期

10 蔣滿貴;家蠶卵酯酶A與滯育性關(guān)系研究進展[J];廣西蠶業(yè);2000年02期

相關(guān)會議論文 前10條

1 黃少虹;江幸福;羅禮智;;昆蟲滯育發(fā)生與調(diào)控的生理生態(tài)機制[A];植物保護與現(xiàn)代農(nóng)業(yè)——中國植物保護學(xué)會2007年學(xué)術(shù)年會論文集[C];2007年

2 肖海軍;何海敏;朱杏芬;薛芳森;;低溫和光周期對黑紋粉蝶滯育解除的影響[A];植�？萍紕�(chuàng)新與病蟲防控專業(yè)化——中國植物保護學(xué)會2011年學(xué)術(shù)年會論文集[C];2011年

3 李秉，

本文編號：1860704