【摘要】：水稻紋枯病(Rice sheath blight)是水稻中重要病害之一,該病害會導(dǎo)致水稻減產(chǎn)和稻米品質(zhì)下降,其致病菌是一類死體營養(yǎng)型真菌--立枯絲核菌(Rhizoctoniasolani)。現(xiàn)階段,雖然發(fā)掘到一些功能性基因?qū)υ摬∮幸种谱饔?但由于水稻抗紋枯病種質(zhì)資源缺乏,因此對該病菌的治理方法仍然以農(nóng)藥防治為主�；谶@種現(xiàn)狀,確有必要開展水稻抵抗紋枯病菌防御反應(yīng)的分子機制研究,獲得更多的抗性種質(zhì)資源,從而為分子育種、提高水稻產(chǎn)量和改善稻米品質(zhì)提供數(shù)據(jù)支持和理論基礎(chǔ)。本研究通過細胞學(xué)觀察、高通量測序和轉(zhuǎn)基因技術(shù)對紋枯病菌侵染后水稻葉鞘組織及其對照之間的細胞變化、基因表達和miRNAs表達變化進行了比較系統(tǒng)的分析,以期探索侵染過程中水稻體內(nèi)發(fā)生的一系列生物學(xué)過程的變化,從而發(fā)現(xiàn)參與防御反應(yīng)的調(diào)控因子和功能基因。本研究主要實驗結(jié)果歸納如下:1.對被侵染的水稻葉鞘組織及其對照的細胞學(xué)觀察發(fā)現(xiàn),侵染24 h后的組織沒有明顯的病斑,且大多數(shù)細胞未被損壞。而侵染48 h后出現(xiàn)明顯的灰褐色病斑,并且染色發(fā)現(xiàn),大面積細胞死亡,說明紋枯病在一定階段大爆發(fā),導(dǎo)致病情急劇加重。2.基于轉(zhuǎn)錄組測序的結(jié)果分析顯示,總共獲得了 430,794,898 clean reads,經(jīng)差異分析表明在24 hpi(hours post-inoculation)和48 hpi階段分別得到了 742和2,825個顯著性差異表達的基因,其中包括一系列與茉莉酸信號、乙烯信號、病程相關(guān)蛋白基因和轉(zhuǎn)錄因子相關(guān)的基因。與茉莉酸和乙烯相關(guān)的差異基因主要參與激素或其衍生物的生物合成和信號轉(zhuǎn)導(dǎo)。其中與茉莉酸合成和信號轉(zhuǎn)導(dǎo)相關(guān)的基因都上調(diào)表達,可能茉莉酸響應(yīng)紋枯病菌誘導(dǎo)含量上升,同時激活下游相關(guān)防御反應(yīng)。差異表達的病程相關(guān)基因可分為12個家族,且大多數(shù)基因呈上調(diào)表達。GO功能富集分析結(jié)果顯示這些基因可能是在細胞質(zhì)行使抑制菌絲侵染的功能。另外,MYB、ERF和WRKY轉(zhuǎn)錄因子家族在調(diào)控水稻防御反應(yīng)中起主要作用,其中WRKY家族基因都上調(diào)表達,推測可能在響應(yīng)紋枯病菌過程中具有調(diào)控特異性。根據(jù)功能和通路分析,不管在24 hpi還是48hpi階段,有許多次級代謝合成相關(guān)的通路可能參與防御紋枯病菌。而且,轉(zhuǎn)錄因子可能在茉莉酸和乙烯激素,與代謝物和病程相關(guān)基因的誘導(dǎo)表達之間起著重要的信號轉(zhuǎn)導(dǎo)作用。3.基于small RNAs測序的結(jié)果分析顯示,在24 hpi和48 hpi中分別獲得了33個差異表達顯著的miRNAs,且所有的差異表達miRNAs總體呈現(xiàn)一種下調(diào)趨勢。之后,從這些差異表達miRNAs數(shù)據(jù)中分別預(yù)測得到195和163個靶標基因。結(jié)合轉(zhuǎn)錄組數(shù)據(jù)分析靶標的表達譜,發(fā)現(xiàn)約一半的靶基因與miRNA的表達呈現(xiàn)負相關(guān)。使用qRT-PCR驗證了部分差異表達miRNAs和相應(yīng)的靶標的表達變化,結(jié)果顯示與測序結(jié)果基本一致。功能分析顯示,這些靶標基因可能主要參與次級代謝通路、脅迫反應(yīng)和植物激素信號轉(zhuǎn)導(dǎo)。4.基于轉(zhuǎn)基因技術(shù)的功能研究結(jié)果顯示,總共獲得了 35株陽性轉(zhuǎn)基因植株,陽性率為70%。在水稻中過量表達osa-miR159b前體序列會顯著性增加osa-miR159b的表達量,以及顯著性下調(diào)靶標基因LOCOs01g59660的表達�？剐澡b定實驗結(jié)果表明,野生型水稻植株(WT)的發(fā)病面積和病情指數(shù)顯著性大于轉(zhuǎn)基因植株(#43和#86),說明osa-miR159b的表達量增加有助于增強水稻對紋枯病菌抗性。5.基于未注釋的miRNAs序列并結(jié)合生物信息學(xué)軟件的結(jié)果分析顯示,篩選獲得了 127個新miRNAs,并對其中17個新miRNAs做了克隆測序鑒定。同時,在已知miRNAs前體序列上,篩選出約80個miRNA*或變體序列,并對osa-miR529*variant 和 osa-miR444c.1*variant 進行了驗證。從新 miRNAs 序列中預(yù)測得到141個靶標基因,并利用5' RACE和瞬時轉(zhuǎn)化方法鑒定了 osa-NmiR1的靶標基因為LOC_Os09g34900,是一個葉綠體轉(zhuǎn)運蛋白,推測可能參與次級代謝物合成。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S435.111.42

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