本文選題：水稻　切入點：稻瘟病　出處：《南京大學》2017年博士論文　論文類型：學位論文

【摘要】：水稻是世界上重要的主糧作物之一,具有悠久的種植歷史和分布很廣的種植區(qū)域。然而,自然界中各種各樣的病原菌不斷地侵襲著水稻,威脅著水稻的安全生產(chǎn)。其中,生理小種多樣且變異速度很快的稻瘟病是水稻所面臨的最嚴重的病害之一。稻瘟病具有分布范圍廣、發(fā)病時期多和病癥后果重等特點,是我國水稻產(chǎn)量嚴重損失的主要原因之一。而目前農(nóng)業(yè)生產(chǎn)上防治稻瘟病的最經(jīng)濟最有效的方法是利用水稻自身所具有的稻瘟病抗性基因進行抗性品種的培育。然而,傳統(tǒng)的抗病育種方法和抗病基因克隆技術都具有費時費力的弊端,已經(jīng)難以滿足當下稻瘟病抗性育種和抗性基因克隆的需求。隨著育成抗病品種的抗性衰減,稻瘟病抗病基因的獲取也逐漸困難,農(nóng)業(yè)生產(chǎn)上可用的抗源日益匱乏。這一系列問題成為當前水稻稻瘟病抗性育種的瓶頸。在先前的研究中,為解決這一問題,本實驗室基于分子進化理論和抗病基因的遺傳變異規(guī)律,提出水稻NBS-LRR類抗病基因中快速演化的位點是對抗快速變異的稻瘟病菌的關鍵所在這一推斷�；诖送茢�,我們成功定位了一系列具有快速演化特點的NBS-LRR位點,并初步驗證了其中一些代表性位點所具有的抗性能力,因而成功建立了一套水稻稻瘟病抗性基因的高通量克隆技術體系。該技術可以實現(xiàn)在較短時間內(nèi)獲取稻瘟病抗性基因,而在很大程度上緩解了稻瘟病抗性基因資源匱乏的問題。本研究在此基礎上,進一步選擇了 4個快速演化位點和2個非快速演化位點,并在稻瘟病抗性水稻材料中進行目的基因的克隆和抗性鑒定研究,最終發(fā)現(xiàn)從4個快速演化位點克隆的9個基因中有8個(～89%)基因表現(xiàn)出一定的稻瘟病抗性,而2個非快速演化位點中克隆的4個基因中未發(fā)現(xiàn)有稻瘟病抗性。該結(jié)果進一步說明了基于快速演化位點的稻瘟病抗病基因高通量克隆技術的有效性和可行性。此外,對快速演化位點和非快速演化位點的基因進行序列分析發(fā)現(xiàn),基因編碼區(qū)的高核苷酸多態(tài)性和高非同義突變率是快速演化位點較好抗性能力的重要基礎。還有基因編碼區(qū)的插入/缺失、mRNA剪切方式變化、NBS區(qū)域重要motif的氨基酸變異以及LRR core數(shù)量變化都是快速演化位點所具有的顯著的遺傳特點�？共』蚝塑账岫鄳B(tài)性傾向的序列特點及其遺傳演化規(guī)律啟示我們可以通過對快速演化位點進行人工改造的方式獲取具有豐富遺傳差異的多樣性的抗病基因�；谶@種啟發(fā),本研究接下來又利用合成生物學技術和基因人工進化技術等分子生物學技術,構建了基于"分段克隆"、"體外重組"和"逐級組裝"這一策略的抗稻瘟病基因的人工進化技術體系。本研究選擇了三個快速演化的稻瘟病抗性位點——Pi37、Pi9與4C134922和兩個非快速演化位點Os05g40150與Os12g17410共計五個位點作為改造靶點,分別經(jīng)過序列篩選、載體構建、分段克隆、人工誘變、體外重組和序列拼裝等過程成功獲取了各靶標基因的重組體。目前,本研究已初步獲得885個陽性克隆(已保留重組DNA材料和載體-重組DNA連接材料,可很快進行大量陽性克隆的獲取),對其中368個進行了 LRR部分重組區(qū)域進行了測序分析,序列分析表明各合成基因間皆存在一定的遺傳差異,且其中絕大部分來自于供體序列中的已有變異。該結(jié)果表明人工體外重組實驗獲得了成功。此外,在對107個載體的稻瘟病抗性鑒定中,我們發(fā)現(xiàn)其中44個具有一定的稻瘟病抗性,某些基因還表現(xiàn)出較強的稻瘟病抗性能力。這一結(jié)果進一步證明了本研究所構建技術體系的可行性與高效性,展現(xiàn)了該技術體系潛在的應用價值。該技術體系不僅可以極大地拓展稻瘟病抗性資源庫,為稻瘟病抗性基因的持續(xù)獲取提供了一條捷徑,具有很好的生產(chǎn)應用前景,還為水稻抗病基因庫的建立和高效利用提供了理論指導和技術支持,同時還為植物抗病基因的理論研究提供了重要的材料。
[Abstract]:Rice is one of the world's most important staple crops, the planting area has long history of cultivation and distribution is very wide. However, a variety of pathogenic bacteria in nature constantly invasion of rice, threatening the safe production of rice. The physiological races of rice blast fungus diversity and variation quickly is one of the most serious rice diseases faced. Rice blast has wide distribution, characteristics of onset period and consequences of heavy illness, is one of the main reasons for China's rice yield serious losses. And at present the method of the most economical and effective agricultural production on the control of rice blast resistant varieties by cultivation of rice blast resistance gene has its own the. However, breeding methods and disease resistance gene cloning technique has drawbacks of traditional time-consuming, has been difficult to meet the current cloning of rice blast resistance breeding and resistance gene With the growing demand. The resistance attenuation of resistant cultivars, obtain the blast resistance gene has been difficult, the agricultural production of resistant sources available is increasingly scarce. These problems become the bottleneck of rice blast resistance breeding. In previous studies, in order to solve this problem, the laboratory of genetic variation in molecular evolution the theory and the resistance gene based on the proposed site of the rapid evolution of rice NBS-LRR resistance gene is the key against the rapid variation of rice blast fungus in this inference. Based on this, we successfully positioned with a series of rapid evolution characteristics of NBS-LRR sites, and a preliminary validation of the resistance of some representative sites has successfully established the technical system, and thus a high-throughput cloning of rice blast resistance genes. This technique can realize the acquisition of rice blast resistance base in a relatively short period of time Because of, but to a large extent eased the problem of shortage of rice blast resistance gene. On this basis, further selected 4 fast evolving sites and 2 non fast evolving sites, and gene cloning and identification of resistance to rice blast resistant rice materials, finally found from the 4 fast the evolution of cloning sites of 9 genes in 8 (~ 89%) gene showed certain resistance to rice blast, and 2 non rapid evolution of 4 loci in the clone was found in rice blast resistance. The results demonstrate the effectiveness of rice blast resistance gene of high-throughput cloning technology fast evolving sites and based on the feasibility. In addition, the sequence analysis found on the site and the rapid evolution of non rapid evolution of genes, the gene encoding high nucleotide polymorphism and high nonsynonymous mutation rate is the rapid evolution of a good site An important basis for resistance. And the gene encoding the insertion / deletion, change of mRNA shear mode, the number of amino acid mutation and LRR core change NBS motif is an important area of the rapid evolution of the genetic characteristics of significant loci. Genetic resistance gene sequence characteristics and nucleotide polymorphism tendency evolution of enlightenment we can acquire resistance gene have abundant diversity of genetic differences through artificial modification of the rapid evolution of sites. Based on this heuristic, this study then use technology and gene technology in artificial synthetic biology and other molecular biology techniques, based on "sub clone", "artificial evolution in vitro recombination system" and "step by step anti assembling" the rice blast resistance gene of this strategy. This study selected three of the rapid evolution of rice blast resistance loci Pi37, Pi9 and 4C134922 and two A non rapid evolution of Os05g40150 and Os12g17410 loci in a total of five sites as a transformation target, respectively after sequence selection, vector construction, sub cloning, artificial mutation, recombination and sequence assembly process successfully obtained the target gene recombinant. At present, this study has preliminarily obtained 885 positive clones (recombinant DNA has been retained material and carrier of recombinant DNA can quickly obtain connection materials, a lot of positive clones), of which 368 were part of the LRR recombination region were sequenced and the sequence analysis showed that the synthetic genes are kept in some genetic differences, and most of them have variation from the donor in the sequence of the. The results showed that the artificial recombination in vitro experiment was successful. In addition, in the identification of rice blast resistance of 107 carriers, we found 44 with blast resistance of certain genes Also showed stronger resistance to blast. This result further proves the feasibility of constructing the system of technology and efficiency, to show the potential application value of the technology system. This technology system not only can greatly expand the blast resistance resource library, provides a shortcut for continued access to rice blast disease resistance gene, has good application prospects, but also provide theoretical guidance and technical support for the establishment of rice disease resistance gene pool and efficient use, but also provides important material for the research of plant disease resistance genes.

【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S435.11

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本文編號：1607606