本文關(guān)鍵詞： 核盤菌 菌核發(fā)育 miRNA 肉桂醇脫氫酶 NADPH氧化酶 ROS　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：核盤菌(Sclerotinia sclerotiorum)是一種在世界廣泛分布的死體營養(yǎng)型病原真菌,因寄主范圍廣且危害大而嚴(yán)重威脅多種重要經(jīng)濟(jì)作物的生產(chǎn)。菌核作為核盤菌重要的初侵染源和抗逆組織,一直是防治菌核病的研究重點(diǎn)和難點(diǎn),從分子水平上剖析核盤菌菌核形成的機(jī)理和影響要素,可以為菌核病害的控制提供新的線索。本研究試圖從mi RNA調(diào)控作用和基因表達(dá)譜分析兩方面入手拓展核盤菌菌核形成機(jī)理的解析。mi RNAs是一類長度在22-24 nt的非編碼小RNA,在動(dòng)物和植物中都發(fā)揮著重要的調(diào)控作用,近幾年在真菌中也發(fā)現(xiàn)有mil RNAs的存在。前期研究中證實(shí)核盤菌也擁有完整的RNAi機(jī)制,可以通過基因沉默手段對目的基因的功能進(jìn)行研究,參與mi RNAs加工合成途徑的關(guān)鍵蛋白酶類Ss-DCL1、Ss-DCL2和Ss-AGO的編碼基因的表達(dá)動(dòng)態(tài)都十分穩(wěn)定,基于此我們推測在核盤菌中也存在mi RNA。本研究利用高通量測序技術(shù)對核盤菌的菌絲生長、菌核發(fā)育、菌核萌發(fā)和侵染致病這4個(gè)重要的生理階段的樣品進(jìn)行測序分析,總計(jì)讀取了13229401條高質(zhì)量的序列,包括922809條特異的s RNAs序列。利用生物信息學(xué)預(yù)測軟件和生化實(shí)驗(yàn)方法,最終我們在核盤菌中鑒定出2個(gè)mil RNAs和42個(gè)候選mil RNAs,它們之間的表達(dá)量差異較大。對一些測序讀數(shù)較高的mil RNAs和候選mil RNAs分子,利用Northern雜交技術(shù)和QRT-PCR方法檢測它們在核盤菌不同生長階段的表達(dá)水平,發(fā)現(xiàn)大多數(shù)mil RNAs在菌絲培養(yǎng)72 h后表達(dá)量有明顯的上調(diào),隨后在菌核成熟階段又顯著回落,這一結(jié)果預(yù)示著相關(guān)mil RNAs可能參與調(diào)控菌核發(fā)育進(jìn)程,另外有幾個(gè)mil RNAs可能還在致病侵染過程中發(fā)揮相應(yīng)作用,需要進(jìn)一步研究分析。為了深入探索核盤菌菌核發(fā)育的分子機(jī)理,我們通過Solexa測序技術(shù)獲得了核盤菌在不同生長發(fā)育階段的基因表達(dá)譜,發(fā)現(xiàn)一個(gè)在菌核形成階段表達(dá)量顯著上調(diào)的基因Ss-CAD(SS1G_10803),推測該基因與核盤菌菌核發(fā)育相關(guān)。本研究通過QRT-PCR技術(shù)進(jìn)一步明確了該基因在菌核形成起始階段表達(dá)量達(dá)到高峰,經(jīng)氨基酸序列比對分析發(fā)現(xiàn)該基因編碼的蛋白屬于肉桂醇脫氫酶(CAD)家族,含有傳統(tǒng)CAD中典型的保守結(jié)構(gòu)域及關(guān)鍵的氨基酸殘基,具有典型的CAD酶活性。核盤菌中Ss-CAD被高效沉默以后,CAD酶活性大幅下降,沉默轉(zhuǎn)化子的菌核發(fā)育起始階段明顯推遲,在胡蘿卜培養(yǎng)基上形成的菌核數(shù)量明顯減少,同時(shí)菌核體積變小,但Ss-CAD沉默對核盤菌的菌絲生長階段及致病力無明顯影響。Ss-CAD沉默對核盤菌菌核發(fā)育的影響與Ss-Nox沉默的情況接近,QRT-PCR結(jié)果表明Ss-CAD沉默后核盤菌中參與ROS生成的基因Ss-Nox1、Ss-Nox2及Ss-Nox R和下游參與ROS代謝的基因Ss-SOD及Ss-CAT的表達(dá)量均有所下降,經(jīng)NBT染色也證實(shí)Ss-CAD沉默后ROS的產(chǎn)量減少。進(jìn)一步研究發(fā)現(xiàn)Ss-CAD沉默菌株對H2O2的耐受能力有所增強(qiáng),在Ss-CAD沉默轉(zhuǎn)化子中超量表達(dá)Ss-Nox1后菌核發(fā)育能力得到了明顯的恢復(fù),而且外源施加6 m M的H2O2或500μM的甲萘醌也有相似恢復(fù)效果。另外在Ss-CAD沉默菌株中,參與NADH和NADPH兩大輔酶系統(tǒng)轉(zhuǎn)換的轉(zhuǎn)氫酶Ss-transhydrogenase的表達(dá)量也顯著下降,NADPH是Ss-Nox產(chǎn)生ROS過程中的重要且唯一的輔酶,外源補(bǔ)充適量NADPH也對菌核發(fā)育有很好的恢復(fù)作用,但NADH和NADP+卻沒有效果。這些結(jié)果均預(yù)示Ss-CAD可能通過影響Nox代謝途徑產(chǎn)生的ROS來影響核盤菌的菌核發(fā)育。
[Abstract]:Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a widely distributed in the world of the necrotrophic fungal pathogen, with wide host range and great harm and serious threat to many economically important crops. The production of sclerotia of Sclerotinia sclerotiorum as primary important infection sources and resistance organization, has been the research focus and difficult treatment against Sclerotinia sclerotiorum the analysis of the sclerotia formation mechanism and influencing factors from the molecular level and provide new clues for sclerotium disease control. This research attempts from the MI RNA regulation and gene expression profiling analysis of two aspects of expanding nuclear disc bacteria nucleus formation mechanism analysis of the.Mi RNAs is a class of 22-24 NT in length non small RNA encoding, can play an important role in animal and plant in recent years in fungi is also found in the presence of RNAs. Mil confirmed ep-1pn also has a RNAi complete mechanism of previous study, through Study on the function of gene silencing means of target gene, Ss-DCL1 key protease synthesis pathway involved in MI RNAs processing, dynamic expression of genes encoding Ss-DCL2 and Ss-AGO are very stable, based on this we speculate that MI RNA. using high-throughput sequencing technology on mycelial growth of S.sclerotiorum, sclerotial development in Sclerotinia bacteria, sclerotium germination and pathogenicity of these 4 important physiological stages were sequenced, total read 13229401 high quality sequences, s RNAs sequence includes 922809 specific. Forecasting software and biochemical experiments using the method of bioinformatics, we end up in S.sclerotiorum identified 2 mil RNAs and 42 candidate mil RNAs, the expression of the differences between them. Some of the larger reads high mil RNAs and mil RNAs candidate molecules by Northern hybridization and QRT-PCR detection method for it The expression level in S.sclerotiorum in different growth stages, found that most mil RNAs in mycelia after 72 h expression were significantly up-regulated and then in mature stage and sclerotium dropped significantly, this result indicates that mil RNAs may be involved in the regulation of sclerotial development process, and several mil RNAs may play a corresponding role in the process of pathogenic infection, need further research and analysis. In order to further explore the sclerotia development molecular mechanism, we obtained gene by Solexa sequencing technology ep-1pn at different developmental stages in the expression profile, found a sclerotium formation stage expression was up-regulated gene Ss-CAD (SS1G_10803), speculated that the gene and nuclear sclerotiorum sclerotial development. This study by QRT-PCR technologies to further clarify the gene expression during the initial stages of Sclerotium formation reached the peak, the amino acid sequence than on Analysis showed that the gene encoding protein belongs to cinnamyl alcohol dehydrogenase (CAD) family, with traditional CAD typical conserved domain and key amino acid residues, with CAD enzyme activity. Typical Sclerotinia Ss-CAD was efficiently silence CAD activity, fell sharply, turn silence son sclerotial development initial stage in cultured carrot was postponed, the number of sclerotia formed on the base was significantly reduced, while the sclerotium of smaller size, but the mycelium of Ss-CAD silencing on Sclerotinia growth stage and pathogenicity had no obvious effect of.Ss-CAD knockdown on sclerotia of Sclerotinia sclerotiorum development influence and Ss-Nox silencing close to QRT-PCR results show that the Ss-Nox1 gene involved in making ROS Ss-CAD silencing sclerotium, expression of gene Ss-SOD and Ss-CAT Ss-Nox2 and Ss-Nox R and ROS in the downstream metabolism decreased, after NBT staining confirmed that ROS reduced the yield of Ss-CAD after the silence. Further studies showed that Ss-CAD silencing strains tolerance to H2O2 increased in Ss-CAD silencing transformants overexpressing Ss-Nox1 after sclerotial development capacity has been significantly restored, and exogenous M 6 M H2O2 or 500 M menadione have similar recovery effect. Also in the silence of Ss-CAD strains, involved in expression the amount of NADH and NADPH conversion of two coenzyme system transhydrogenase Ss-transhydrogenase also significantly decreased, NADPH Ss-Nox is the ROS process produced an important and unique supplement of exogenous coenzyme NADPH also has a good recovery effect on sclerotial development, but NADH and NADP+ had no effect. These results indicate Ss-CAD may be produced by effect of Nox ROS metabolic pathway to influence Sclerotinia sclerotium development.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S432.44

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本文編號(hào)：1537363