核盘菌中milRNA的预测和验证以及肉桂醇脱氢酶编码基因的功能研究

发布时间：2018-02-26 09:04

本文关键词： 核盘菌菌核发育 miRNA 肉桂醇脱氢酶 NADPH氧化酶 ROS　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：核盘菌(Sclerotinia sclerotiorum)是一种在世界广泛分布的死体营养型病原真菌,因寄主范围广且危害大而严重威胁多种重要经济作物的生产。菌核作为核盘菌重要的初侵染源和抗逆组织,一直是防治菌核病的研究重点和难点,从分子水平上剖析核盘菌菌核形成的机理和影响要素,可以为菌核病害的控制提供新的线索。本研究试图从mi RNA调控作用和基因表达谱分析两方面入手拓展核盘菌菌核形成机理的解析。mi RNAs是一类长度在22-24 nt的非编码小RNA,在动物和植物中都发挥着重要的调控作用,近几年在真菌中也发现有mil RNAs的存在。前期研究中证实核盘菌也拥有完整的RNAi机制,可以通过基因沉默手段对目的基因的功能进行研究,参与mi RNAs加工合成途径的关键蛋白酶类Ss-DCL1、Ss-DCL2和Ss-AGO的编码基因的表达动态都十分稳定,基于此我们推测在核盘菌中也存在mi RNA。本研究利用高通量测序技术对核盘菌的菌丝生长、菌核发育、菌核萌发和侵染致病这4个重要的生理阶段的样品进行测序分析,总计读取了13229401条高质量的序列,包括922809条特异的s RNAs序列。利用生物信息学预测软件和生化实验方法,最终我们在核盘菌中鉴定出2个mil RNAs和42个候选mil RNAs,它们之间的表达量差异较大。对一些测序读数较高的mil RNAs和候选mil RNAs分子,利用Northern杂交技术和QRT-PCR方法检测它们在核盘菌不同生长阶段的表达水平,发现大多数mil RNAs在菌丝培养72 h后表达量有明显的上调,随后在菌核成熟阶段又显著回落,这一结果预示着相关mil RNAs可能参与调控菌核发育进程,另外有几个mil RNAs可能还在致病侵染过程中发挥相应作用,需要进一步研究分析。为了深入探索核盘菌菌核发育的分子机理,我们通过Solexa测序技术获得了核盘菌在不同生长发育阶段的基因表达谱,发现一个在菌核形成阶段表达量显著上调的基因Ss-CAD(SS1G_10803),推测该基因与核盘菌菌核发育相关。本研究通过QRT-PCR技术进一步明确了该基因在菌核形成起始阶段表达量达到高峰,经氨基酸序列比对分析发现该基因编码的蛋白属于肉桂醇脱氢酶(CAD)家族,含有传统CAD中典型的保守结构域及关键的氨基酸残基,具有典型的CAD酶活性。核盘菌中Ss-CAD被高效沉默以后,CAD酶活性大幅下降,沉默转化子的菌核发育起始阶段明显推迟,在胡萝卜培养基上形成的菌核数量明显减少,同时菌核体积变小,但Ss-CAD沉默对核盘菌的菌丝生长阶段及致病力无明显影响。Ss-CAD沉默对核盘菌菌核发育的影响与Ss-Nox沉默的情况接近,QRT-PCR结果表明Ss-CAD沉默后核盘菌中参与ROS生成的基因Ss-Nox1、Ss-Nox2及Ss-Nox R和下游参与ROS代谢的基因Ss-SOD及Ss-CAT的表达量均有所下降,经NBT染色也证实Ss-CAD沉默后ROS的产量减少。进一步研究发现Ss-CAD沉默菌株对H2O2的耐受能力有所增强,在Ss-CAD沉默转化子中超量表达Ss-Nox1后菌核发育能力得到了明显的恢复,而且外源施加6 m M的H2O2或500μM的甲萘醌也有相似恢复效果。另外在Ss-CAD沉默菌株中,参与NADH和NADPH两大辅酶系统转换的转氢酶Ss-transhydrogenase的表达量也显著下降,NADPH是Ss-Nox产生ROS过程中的重要且唯一的辅酶,外源补充适量NADPH也对菌核发育有很好的恢复作用,但NADH和NADP+却没有效果。这些结果均预示Ss-CAD可能通过影响Nox代谢途径产生的ROS来影响核盘菌的菌核发育。
[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.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.44

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