本文關(guān)鍵詞：寄生疫霉菌小RNA多樣性及其參與基因表達調(diào)控的探索研究　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 寄生疫霉菌 本氏煙 小RNA 轉(zhuǎn)錄組 抗病

【摘要】：卵菌是一類形態(tài)上與真菌相似,但進化上和動植物以及真菌關(guān)系較遠的真核微生物。其中疫霉屬(Phytophthora)卵菌的多個病原物在世界范圍內(nèi)引起毀滅性的植物病害,每年造成嚴(yán)重的經(jīng)濟損失。由于進化上的獨特性,卵菌在生理生化和侵染機制上與真菌差異很大,導(dǎo)致許多對真菌有效的殺菌劑對卵菌無效。因此探索疫霉菌-植物互作機制在植物病理學(xué)研究和田間生產(chǎn)實踐中都具有重要的意義。另外,抗病品種的使用在田間病害防控中發(fā)揮著重要的作用,但疫霉菌毒性變異迅速,常通過丟失、突變無毒基因來逃避寄主抗病基因的識別,造成對應(yīng)抗病品種失效。然而一些毒性菌株中的無毒基因尚未發(fā)生任何突變但基因不表達,這說明表觀遺傳學(xué)調(diào)控可能參與調(diào)節(jié)寄生疫霉菌毒性變異�；诖�,我們分析了寄生疫霉菌(P.parasitica)小RNA的特點以及其在沉默內(nèi)源基因尤其是致病相關(guān)基因方面發(fā)揮的功能。同時,為了了解疫霉菌-植物互作機制,我們圍繞寄生疫霉菌-本氏煙模式互作體系,從基因組學(xué)和轉(zhuǎn)錄組學(xué)角度分析寄生疫霉菌與寄主植物親和互作的分子機制。主要研究結(jié)果如下:1.通過高通量測序技術(shù)對寄生疫霉菌小RNA多樣性的研究發(fā)現(xiàn):(a)寄生疫霉菌累積大量的25-26nt和少量21nt小RNA,同時也累積一系列侵染階段高度誘導(dǎo)表達的29nt和33nt的tRNA來源的小RNA。25-26nt小RNA和21nt小RNA具有強烈的5'堿基偏好性,無鏈特異性地成簇分布在基因組上。(b)超過7000個內(nèi)源基因位點累積25-26nt小RNA,轉(zhuǎn)錄組測序表明這些基因一般在營養(yǎng)生長階段和侵染階段均不表達,qPCR實驗支持轉(zhuǎn)錄組測序的結(jié)果。小RNA在外顯子、內(nèi)含子以及基因上下游區(qū)域均勻累積,說明基因沉默可能發(fā)生在轉(zhuǎn)錄水平。(c)累積25-26nt小RNA的基因主要為寄生疫霉菌特有的基因,包含大約40%的RXLR效應(yīng)基因、50%的Crinkler效應(yīng)基因和一些激發(fā)子基因,說明25-26nt小RNA在新基因沉默和寄生疫霉菌毒性變異中可能發(fā)揮重要作用。(d)25-26nt小RNA主要來源于基因稀疏區(qū)和基因密集區(qū),且小RNA的產(chǎn)生和沉默狀態(tài)能從重復(fù)區(qū)向兩側(cè)傳播幾百堿基到上千堿基距離。與此呼應(yīng),累積小RNA的內(nèi)源基因大部分位于或十分鄰近重復(fù)區(qū)域。(e)21nt小RNA主要來源于高表達基因的外顯子區(qū),其累積不足以導(dǎo)致基因完全沉默,且小RNA的產(chǎn)生可能依賴基因表達。(f)對已公布的致病疫霉菌的小RNA和轉(zhuǎn)錄組數(shù)據(jù)重新分析的結(jié)果表明,致病疫霉菌中25-26nt小RNA,而不是21nt小RNA,也與內(nèi)源基因的沉默密切相關(guān),這說明這一現(xiàn)象在疫霉菌中是保守的。2.通過對寄生疫霉菌侵染本氏煙(Nicotiana benthamiana)離體葉片不同時間點(0、3hpi、6hpi、12hpi、24hpi、48hpi)的樣品進行轉(zhuǎn)錄組測序和分析,結(jié)果表明(a)侵染階段早期和晚期基因表達差異很大,大量致病相關(guān)基因在侵染階段早期上調(diào)表達而在晚期下調(diào)表達。基于此,我們鑒定了 231個侵染階段早期特異性高表達的編碼分泌蛋白的基因,這包括大量編碼RXLR效應(yīng)蛋白、NPP樣激發(fā)子以及各種水解酶和蛋白酶抑制劑的基因。同時還發(fā)現(xiàn)編碼鹽酸小檗堿樣蛋白、碳酸酐酶和SCP樣胞外蛋白的三個家族基因以及多個功能未知的基因家族在侵染階段早期特異性高表達。我們圍繞其中一個家族進行系統(tǒng)地分析,發(fā)現(xiàn)其家族成員在疫霉中頻繁復(fù)制且出現(xiàn)顯著的結(jié)構(gòu)域重排現(xiàn)象,根據(jù)結(jié)構(gòu)域排列特點可將其分成三個亞家族。(b)大量蛋白翻譯、核小體組裝以及RNA甲基化相關(guān)基因在侵染早期顯著上調(diào)表達,而三羧酸循環(huán)和ATP合成相關(guān)基因在侵染中后期上調(diào)表達。但與預(yù)期不符的是,囊泡介導(dǎo)的轉(zhuǎn)運、微管介導(dǎo)的細胞運輸、蛋白降解、蛋白磷酸化和信號傳導(dǎo)相關(guān)基因在侵染階段顯著下調(diào)表達。3.此外,我們利用轉(zhuǎn)錄組數(shù)據(jù)對本氏煙基因轉(zhuǎn)錄本進行了組裝和表達分析,并且通過鑒定抗病相關(guān)的基因來探索寄主植物防御疫霉菌的分子機制。結(jié)果表明(a)包括活性氧進發(fā)和程序化細胞壞死相關(guān)基因和PAMP誘導(dǎo)的基因在內(nèi)的大量防御反應(yīng)基因在侵染階段上調(diào)表達。水楊酸、茉莉酸和乙烯的合成和信號通路相關(guān)基因均上調(diào)表達,進一步分析表明這些激素相關(guān)基因以及flg22/e1f18誘導(dǎo)表達的基因絕大部分在侵染階段上調(diào)表達,且在3hpi和24hpi出現(xiàn)兩個表達高峰,這可能是大部分抗病相關(guān)基因的重要特征�；诖�,我們鑒定到了 3766個在3hpi和24hpi出現(xiàn)兩個表達高峰的基因作為候選的抗病相關(guān)基因,其中753個在3hpi上調(diào)超過10倍。(b)系統(tǒng)地鑒定了本氏煙中的蛋白激酶和轉(zhuǎn)錄因子,它們大部分也在3hpi和24hpi出現(xiàn)兩個表達高峰。我們進一步鑒定到了在3hpi和24hpi出現(xiàn)兩個表達高峰的一系列受體樣蛋白激酶和4個受體樣蛋白,為PAMP/激發(fā)子受體篩選提供了重要的候選。(d)大量蛋白分泌、蛋白修飾以及包括糖異生、脂肪酸氧化以及三羧酸循環(huán)在內(nèi)的分解代謝過程相關(guān)基因在侵染階段顯著上調(diào)表達;而以蛋白翻譯、脂肪酸和糖類合成為代表的大量生物合成相關(guān)通路、光合作用以及離子轉(zhuǎn)運等基礎(chǔ)代謝過程相關(guān)基因在侵染階段顯著下調(diào)表達。
[Abstract]:The egg is a kind of form of bacteria and fungi are similar, but the evolution of eukaryotic microorganisms and plants and animals and fungi. Among distantly related Phytophthora (Phytophthora) multiple pathogens caused by the oomycete plant disease of destruction in the world, causing serious economic losses every year. By the unique evolution the differences in physiological and biochemical and fungal infection mechanism of large egg bacteria, causing many fungi on the sterilization of effective agent against oomycete Phytophthora is invalid. Therefore exploring plant interaction mechanism has important significance in the research and production practice in the field of plant pathology. In addition, the use of resistant varieties of play an important role in the field of disease prevention and control, but the toxicity of Phytophthora mutate rapidly, often through the loss of avirulence gene mutations to evade the host recognition of resistance genes and resistant varieties. However, failure caused by the corresponding toxicity in some strains of non-toxic base Because there is not any mutation but the genes are not expressed, indicating that epigenetic regulation may be involved in the regulation of Virulence Variation of Phytophthora parasitica. Based on this, we analyzed the parasitic Phytophthora (P.parasitica) characteristics of RNA and its endogenous gene in silence especially pathogenic related gene function. At the same time, in order to understand the interaction the mechanism of Phytophthora plants, we focus on Phytophthora parasitica - benthamiana mode interaction system, from the molecular mechanism analysis of Phytophthora parasitica and host plant compatible interaction point of genomics and transcriptomics. The main results are as follows: 1. by high-throughput sequencing of Phytophthora parasitica RNA diversity research: (a) Phytophthora parasitica accumulate a large amount of 25-26nt and a little 21nt RNA, but also accumulated a series of infection stage height to induce the expression of 29nt and 33nt from tRNA and 21nt small RNA.25-26nt small RNA RNA has a small 5'base strong preference, no chain specifically clustered in genome. (b) more than 7000 endogenous gene loci and accumulation of 25-26nt small RNA transcriptome sequencing showed that these genes generally in the vegetative growth stage and infection stage were not expressed and qPCR experimental support for transcriptome sequencing of small RNA results. In exon, Chi Ko and gene on the downstream area of uniform accumulation, that gene silencing may occur at the transcriptional level. (c) the cumulative 25-26nt small RNA genes were mainly endemic parasitic Phytophthora genes, including the RXLR effect about 40% genes, 50% genes of Crinkler effect and some elicitor gene, 25-26nt small RNA may play an important role in the new gene silencing and parasitic Phytophthora virulence variation. (d) 25-26nt RNA gene mainly comes from sparse areas and dense areas of genes, and small RNA generation and silence from the repeat region To spread to hundreds of thousands of bases on both sides of the base distance. And this ECHO, endogenous gene RNA located mostly in the accumulation of small or very adjacent area. (E) 21nt RNA from high expression gene, its accumulation is not enough to cause gene silence, and produced a small RNA gene may depend on the expression (f). The re analysis of Phytophthora infestans published little RNA and transcriptome data showed that 25-26nt RNA of Phytophthora infestans, rather than 21nt RNA, and endogenous gene silencing is closely related to the explanation of this phenomenon in Phytophthora is conserved by parasitic.2. the infection of Phytophthora benthamiana (Nicotiana benthamiana) leaves in vitro at different time points (0,3hpi, 6hpi, 12hpi, 24hpi, 48hpi) samples transcriptome sequencing and analysis. The results show that (a) early and late infection stage gene expression differences, a large number of pathogenic Expression of related genes in the early stage of infection in the late expression. Based on this, we identified 231 early infection stage specific expression of genes encoding secreted proteins, which includes a large number of RXLR encoding NPP like effector protein elicitor and various hydrolytic enzymes and protein enzyme inhibitor. It was also found that the gene encoding hydrochloride berberine like protein three gene family of carbonic anhydrase and SCP like extracellular proteins and unknown function gene family in the early infection stage specific expression. We focus on one family system analysis, found that the family members in Phytophthora replication and frequent domain rearrangements significantly. According to the domain arrangement features can be divided into three subfamilies (b). A large number of protein translation, nucleosome assembly and RNA methylation related gene expression was significantly up-regulated in early infection, The expression of three tricarboxylic acid cycle and ATP synthesis related genes in the infection in the late rise. But inconsistent with expectations, vesicle mediated transport, microtubule mediated cell transport, protein degradation, protein phosphorylation and signal transduction related genes significantly down regulated expression of.3. in the infection stage. In addition, we use the transcriptome data of benthamiana gene transcripts were assembled and expression analysis, and through the identification of disease resistance related genes to explore the molecular mechanism of host plant defense of Phytophthora species. The results showed that (a) including a large number of up-regulated gene activity in the oxidative burst and programmed cell necrosis related genes induced by PAMP, and the defense response gene in the infection stage. Salicylic acid, synthesis and signaling pathway related genes in ethylene and jasmonic acid were up-regulated, further analysis showed that these hormone related genes and flg22/e1f18 genes induced by most Expression in the infection stage increases, and two peak expression in 3hpi and 24hpi, which may be an important feature of most genes related to disease resistance. Based on this, we identified 3766 two peak expression genes as candidate genes related to disease resistance in 3hpi and 24hpi, 753 of them in the 3hpi increase more than 10 times. (b) system to identify benthamiana in protein kinases and transcription factors, most of them are 3hpi and 24hpi appeared two peak expression. We further identified to the appearance of the two peak expression of a series of receptor like protein kinase and 4 receptor like protein in 3hpi and 24hpi, provides the important candidate for screening PAMP/ elicitor receptor (d). A large number of proteins, including protein modification and expression of gluconeogenesis, fatty acid oxidation and three tricarboxylic acid cycle, metabolism related genes in the infection stage significantly increased; The related genes such as a large number of biosynthetic pathways, photosynthesis and ion transport were significantly down regulated in protein translation.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S432.4

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9 鄒艷芬;子癇前期中非編碼RNA對滋養(yǎng)細胞功能的調(diào)控及機制探索[D];南京醫(yī)科大學(xué);2015年

10 朱喬;miR-10b在人肝細胞肝癌發(fā)生中的作用及其機制的初步探索[D];第四軍醫(yī)大學(xué);2015年

中國碩士學(xué)位論文全文數(shù)據(jù)庫 前10條

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7 陳瑞東;長鏈非編碼RNA MEG3在胰腺癌發(fā)生發(fā)展中作用的實驗研究[D];蘇州大學(xué);2015年

8 劉駿武;線蟲和水稻中環(huán)狀RNA的預(yù)測及分析[D];華中農(nóng)業(yè)大學(xué);2015年

9 李猷;利用RNA干擾技術(shù)提高番茄抗TMV侵染能力的研究[D];牡丹江師范學(xué)院;2015年

10 吳術(shù)盛;SVCV感染EPC細胞microRNA表達譜的初步研究[D];華中農(nóng)業(yè)大學(xué);2015年

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