响应纹枯病菌的水稻基因及其miRNA的表达分析与功能研究

发布时间：2018-09-03 08:14

【摘要】：水稻纹枯病(Rice sheath blight)是水稻中重要病害之一,该病害会导致水稻减产和稻米品质下降,其致病菌是一类死体营养型真菌--立枯丝核菌(Rhizoctoniasolani)。现阶段,虽然发掘到一些功能性基因对该病有抑制作用,但由于水稻抗纹枯病种质资源缺乏,因此对该病菌的治理方法仍然以农药防治为主。基于这种现状,确有必要开展水稻抵抗纹枯病菌防御反应的分子机制研究,获得更多的抗性种质资源,从而为分子育种、提高水稻产量和改善稻米品质提供数据支持和理论基础。本研究通过细胞学观察、高通量测序和转基因技术对纹枯病菌侵染后水稻叶鞘组织及其对照之间的细胞变化、基因表达和miRNAs表达变化进行了比较系统的分析,以期探索侵染过程中水稻体内发生的一系列生物学过程的变化,从而发现参与防御反应的调控因子和功能基因。本研究主要实验结果归纳如下:1.对被侵染的水稻叶鞘组织及其对照的细胞学观察发现,侵染24 h后的组织没有明显的病斑,且大多数细胞未被损坏。而侵染48 h后出现明显的灰褐色病斑,并且染色发现,大面积细胞死亡,说明纹枯病在一定阶段大爆发,导致病情急剧加重。2.基于转录组测序的结果分析显示,总共获得了 430,794,898 clean reads,经差异分析表明在24 hpi(hours post-inoculation)和48 hpi阶段分别得到了 742和2,825个显著性差异表达的基因,其中包括一系列与茉莉酸信号、乙烯信号、病程相关蛋白基因和转录因子相关的基因。与茉莉酸和乙烯相关的差异基因主要参与激素或其衍生物的生物合成和信号转导。其中与茉莉酸合成和信号转导相关的基因都上调表达,可能茉莉酸响应纹枯病菌诱导含量上升,同时激活下游相关防御反应。差异表达的病程相关基因可分为12个家族,且大多数基因呈上调表达。GO功能富集分析结果显示这些基因可能是在细胞质行使抑制菌丝侵染的功能。另外,MYB、ERF和WRKY转录因子家族在调控水稻防御反应中起主要作用,其中WRKY家族基因都上调表达,推测可能在响应纹枯病菌过程中具有调控特异性。根据功能和通路分析,不管在24 hpi还是48hpi阶段,有许多次级代谢合成相关的通路可能参与防御纹枯病菌。而且,转录因子可能在茉莉酸和乙烯激素,与代谢物和病程相关基因的诱导表达之间起着重要的信号转导作用。3.基于small RNAs测序的结果分析显示,在24 hpi和48 hpi中分别获得了33个差异表达显著的miRNAs,且所有的差异表达miRNAs总体呈现一种下调趋势。之后,从这些差异表达miRNAs数据中分别预测得到195和163个靶标基因。结合转录组数据分析靶标的表达谱,发现约一半的靶基因与miRNA的表达呈现负相关。使用qRT-PCR验证了部分差异表达miRNAs和相应的靶标的表达变化,结果显示与测序结果基本一致。功能分析显示,这些靶标基因可能主要参与次级代谢通路、胁迫反应和植物激素信号转导。4.基于转基因技术的功能研究结果显示,总共获得了 35株阳性转基因植株,阳性率为70%。在水稻中过量表达osa-miR159b前体序列会显著性增加osa-miR159b的表达量,以及显著性下调靶标基因LOCOs01g59660的表达。抗性鉴定实验结果表明,野生型水稻植株(WT)的发病面积和病情指数显著性大于转基因植株(#43和#86),说明osa-miR159b的表达量增加有助于增强水稻对纹枯病菌抗性。5.基于未注释的miRNAs序列并结合生物信息学软件的结果分析显示,筛选获得了 127个新miRNAs,并对其中17个新miRNAs做了克隆测序鉴定。同时,在已知miRNAs前体序列上,筛选出约80个miRNA*或变体序列,并对osa-miR529*variant 和 osa-miR444c.1*variant 进行了验证。从新 miRNAs 序列中预测得到141个靶标基因,并利用5' RACE和瞬时转化方法鉴定了 osa-NmiR1的靶标基因为LOC_Os09g34900,是一个叶绿体转运蛋白,推测可能参与次级代谢物合成。
[Abstract]:Rice sheath blight is one of the most important rice diseases. It can lead to rice yield reduction and rice quality degradation. The pathogen is a kind of dead vegetative fungi, Rhizoctonia solani. At present, although some functional genes have been found to inhibit the disease, the resistance to rice sheath blight is due to the resistance of rice varieties to rice sheath blight. Based on this situation, it is necessary to study the molecular mechanism of rice resistance to sheath blight and obtain more resistant germplasm resources, so as to provide data support and theoretical basis for molecular breeding, increasing rice yield and improving rice quality. Basis. In this study, we systematically analyzed the cell changes, gene expression and microRNAs expression in rice sheath tissues and control tissues infected by Rhizoctonia solani by cytological observation, high-throughput sequencing and transgenic technology, in order to explore the changes of a series of biological processes occurring in Rice during the process of infection, and consequently the occurrence of the disease. The main results of this study were summarized as follows: 1. Cytological observation of infected rice leaf sheath tissue and its control showed that there were no obvious lesions in the tissues 24 hours after infection, and most of the cells were not damaged. The results of transcriptome sequencing analysis showed that 430,794,898 clean reads were obtained, and 742 and 2,825 significant differential expressions were obtained at 24 HPI (hours post-inoculation) and 48 HPI stages, respectively. Genes, including a series of genes associated with jasmonic acid signaling, ethylene signaling, disease-related protein genes and transcription factors. Differential genes associated with jasmonic acid and ethylene are involved in the biosynthesis and signal transduction of hormones or their derivatives. Genes associated with jasmonic acid synthesis and signal transduction are up-regulated. The differentially expressed pathogenesis-related genes can be divided into 12 families, and most of them are up-regulated. GO enrichment analysis showed that these genes may play a role in inhibiting mycelial infection in cytoplasm. In addition, MYB, ERF and WRKY transfection may be involved. The transcription factor family plays a major role in regulating rice defense responses, and the WRKY family genes are up-regulated, presumably having regulatory specificity in response to Rhizoctonia solani. Furthermore, transcription factors may play an important role in signal transduction between jasmonic acid and ethylene hormones, and in inducing the expression of metabolites and disease-related genes. After that, 195 and 163 target genes were predicted from these differentially expressed microRNAs data. By analyzing the target expression profiles, we found that about half of the target genes were negatively correlated with the expression of microRNAs. Functional analysis showed that these target genes may be involved in secondary metabolic pathways, stress responses and plant hormone signaling transduction. 4. Functional studies based on transgenic technology showed that 35 transgenic plants were positive, with a positive rate of 70%. Sequences significantly increased the expression of osa-microRNA159b and significantly decreased the expression of the target gene LOCOs01g59660. Resistance identification results showed that the incidence area and disease index of wild type rice (WT) were significantly greater than those of transgenic rice (# 43 and # 86), suggesting that the increased expression of osa-microRNA159b could help to enhance rice resistance to sheath blight. Bacterial resistance. 5. Based on unannotated microRNAs sequences and combined with bioinformatics software, 127 new microRNAs were screened and 17 of them were cloned and sequenced. * variant was validated. 141 target genes were predicted from the new microRNAs sequence, and the target gene of osa-Nmicro1 was identified as LOC_Os09g34900 by 5'RACE and transient transformation method. It was suggested that osa-Nmicro1 might be involved in secondary metabolite synthesis.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.111.42

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