拟松材线虫种群遗传多样性及其致病相关基因的功能研究

Bursaphelenchus mucronatus is the closest homologs of Bursaphelenchus xylophilus which not only manifested in morphological similarity but also in genetic relationship among all other parasitic nematodes. Recently, more and more evidences were found that B. mucronatus would also contribute to the death of pine trees. Meanwhile, many researches proved that B. mucronatus also had pathogenicity and exhibited pathogenicity differentiation among their species. A large amount of B. mucronatus species will be collected from all over the world and their corresponding genome resource bank will be constructed in this project. Based on this foundation, we employ morphology, molecular biology, population genetics, bio-informatics and statistics to dig out genes related with morphological variation, pathogenicity variation and genetic variation followed with analysis on the function of some pathogenicity gene candidates. That is to say, we attempt to make a comprehensive evaluation on the harmfulness of B. mucronatus and figure out the dynamic law of B. mucronatus morphological variation, genetic variation and pathogenicity differentiation and the relationship among them. In addition, we aim to develop some biomarkers based on morphological and genetics to distinguish the pathogenicity of B. mucronatus and screen out some pathogenic genes. Moreover, we try to shed light on the critical roles that some pathogenic genes played during the pathogenic process and provide useful information on the prevention as well as molecular pathogenic mechanism of B. mucronatus in order to reduce pine trees damage.

拟松材线虫是与松材线虫在形态上以及近缘关系上最亲近的一种松树寄生线虫。近年来生产上出现了越来越多的松树死亡现象与拟松材线虫有关。与此同时，越来越多的研究表明，拟松材线虫有致病性，并且存在致病力分化现象。本课题将在广泛收集国内外拟松材线虫虫株、建立拟松拟松材线虫基因资源库的基础上，综合运用形态学、分子生物学、群体遗传学、基因生物信息学、统计学等原理和方法，开展拟松材线虫种群形态变异、致病性变异、遗传变异、致病性相关候选基因的发掘、致病基因的功能分析等研究工作，从而对拟松材线虫的危害做出总体评价；试图弄清楚在我国境内不同地域分布的拟松材线虫种群之间的形态变异、遗传变异、致病性分化的规律以及三者之间的相关性；开发与致病性相关的形态学标记和遗传学标记；筛选出拟松材线虫致病相关基因；阐明拟松材线虫致病过程中的关键基因及其功能。为有效防控拟松材线虫对松树的危害以及拟松材线虫致病的分子机理提供理论基础。

拟松材线虫是与松材线虫在形态上以及近缘关系上最亲近的一种松树寄生线虫。近年来生产上出现了越来越多的松树死亡现象与拟松材线虫有关。与此同时，越来越多的研究表明，拟松材线虫有致病性，并且存在致病力分化现象。本课题在广泛收集国内外拟松材线虫虫株、建立拟松拟松材线虫基因资源库的基础上，综合运用形态学、分子生物学、群体遗传学、基因生物信息学、统计学等原理和方法，开展了拟松材线虫种群形态变异、致病性变异、遗传变异、致病性相关候选基因的发掘、致病基因的功能分析等研究工作，取得了以下主要成果：① 建立我国首个拟松材线虫基因资源库。以采用活体形式培养了86个拟松材线虫虫株，以DNA形式保存了92个拟松材线虫基因资源；② 进一步证实拟松材线虫对松树的致病性，拟松材线虫能够对松林安全树构成严重威胁。③ 阐明拟松材线虫种群之间的形态变异、遗传变异、致病性分化以及三者之间的相关性。长期的地理隔离，导致拟松材线虫各虫株间的生殖隔离，进而形成不同致病力的拟松材线虫虫株。④ 筛选并获得拟松材线虫致病相关基因。共获得了约40000个基因和5000个蛋白。5个致病相关蛋白在强致病力虫株中都是高表达的，其中过氧化物酶、脂肪酸与视黄醇结合蛋白、谷胱甘肽过氧化物酶与应对寄主防御反应有关，纤维素酶、扩张蛋白与破坏寄主细胞壁有关。⑤阐明了拟松材线虫致病过程中的关键基因及其功能。借助RNAi技术并结合接种实验对拟松材线虫β-1,4-内切葡聚糖酶基因Bm-eng-1、双半胱氨酸过氧化物酶基因2-Cys Prx、脂肪酸与视黄醇结合蛋白（Fatty acid- and retinol-binding protein，FAR）基因、钙网蛋白（Calreticulin，CRT）基因的功能进行研究，这些基因在拟松材线虫致病过程中起着重要作用。项目研究结果为我国有效防控拟松材线虫对松树的危害以及拟松材线虫致病的分子机理提供理论基础。