抗稻痕病基因快速演化位点的遗传分析与人工进化研究

发布时间：2018-03-13 18:20

本文选题：水稻　切入点：稻瘟病　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：水稻是世界上重要的主粮作物之一,具有悠久的种植历史和分布很广的种植区域。然而,自然界中各种各样的病原菌不断地侵袭着水稻,威胁着水稻的安全生产。其中,生理小种多样且变异速度很快的稻瘟病是水稻所面临的最严重的病害之一。稻瘟病具有分布范围广、发病时期多和病症后果重等特点,是我国水稻产量严重损失的主要原因之一。而目前农业生产上防治稻瘟病的最经济最有效的方法是利用水稻自身所具有的稻瘟病抗性基因进行抗性品种的培育。然而,传统的抗病育种方法和抗病基因克隆技术都具有费时费力的弊端,已经难以满足当下稻瘟病抗性育种和抗性基因克隆的需求。随着育成抗病品种的抗性衰减,稻瘟病抗病基因的获取也逐渐困难,农业生产上可用的抗源日益匮乏。这一系列问题成为当前水稻稻瘟病抗性育种的瓶颈。在先前的研究中,为解决这一问题,本实验室基于分子进化理论和抗病基因的遗传变异规律,提出水稻NBS-LRR类抗病基因中快速演化的位点是对抗快速变异的稻瘟病菌的关键所在这一推断。基于此推断,我们成功定位了一系列具有快速演化特点的NBS-LRR位点,并初步验证了其中一些代表性位点所具有的抗性能力,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术体系。该技术可以实现在较短时间内获取稻瘟病抗性基因,而在很大程度上缓解了稻瘟病抗性基因资源匮乏的问题。本研究在此基础上,进一步选择了 4个快速演化位点和2个非快速演化位点,并在稻瘟病抗性水稻材料中进行目的基因的克隆和抗性鉴定研究,最终发现从4个快速演化位点克隆的9个基因中有8个(～89%)基因表现出一定的稻瘟病抗性,而2个非快速演化位点中克隆的4个基因中未发现有稻瘟病抗性。该结果进一步说明了基于快速演化位点的稻瘟病抗病基因高通量克隆技术的有效性和可行性。此外,对快速演化位点和非快速演化位点的基因进行序列分析发现,基因编码区的高核苷酸多态性和高非同义突变率是快速演化位点较好抗性能力的重要基础。还有基因编码区的插入/缺失、mRNA剪切方式变化、NBS区域重要motif的氨基酸变异以及LRR core数量变化都是快速演化位点所具有的显著的遗传特点。抗病基因核苷酸多态性倾向的序列特点及其遗传演化规律启示我们可以通过对快速演化位点进行人工改造的方式获取具有丰富遗传差异的多样性的抗病基因。基于这种启发,本研究接下来又利用合成生物学技术和基因人工进化技术等分子生物学技术,构建了基于"分段克隆"、"体外重组"和"逐级组装"这一策略的抗稻瘟病基因的人工进化技术体系。本研究选择了三个快速演化的稻瘟病抗性位点——Pi37、Pi9与4C134922和两个非快速演化位点Os05g40150与Os12g17410共计五个位点作为改造靶点,分别经过序列筛选、载体构建、分段克隆、人工诱变、体外重组和序列拼装等过程成功获取了各靶标基因的重组体。目前,本研究已初步获得885个阳性克隆(已保留重组DNA材料和载体-重组DNA连接材料,可很快进行大量阳性克隆的获取),对其中368个进行了 LRR部分重组区域进行了测序分析,序列分析表明各合成基因间皆存在一定的遗传差异,且其中绝大部分来自于供体序列中的已有变异。该结果表明人工体外重组实验获得了成功。此外,在对107个载体的稻瘟病抗性鉴定中,我们发现其中44个具有一定的稻瘟病抗性,某些基因还表现出较强的稻瘟病抗性能力。这一结果进一步证明了本研究所构建技术体系的可行性与高效性,展现了该技术体系潜在的应用价值。该技术体系不仅可以极大地拓展稻瘟病抗性资源库,为稻瘟病抗性基因的持续获取提供了一条捷径,具有很好的生产应用前景,还为水稻抗病基因库的建立和高效利用提供了理论指导和技术支持,同时还为植物抗病基因的理论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.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.11

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