【作者】 王颖；

【导师】 张星耀；

【作者基本信息】 东北林业大学 ， 森林保护学， 2014， 博士

【摘要】 松材线虫病(Pine wilt disease)又称松树萎蔫病或松树枯萎病,是松树的一种毁灭性病害。目前已知分布于中国、日本、韩国、美国、加拿大、墨西哥、葡萄牙、西班牙等国家。我国自上世纪80年代初首次发现该病以来,病情扩散蔓延迅速,已对我国的松林生态系统构成了严重的破坏和威胁,成为我国最危险的森林有害生物之一。该病害的发生涉及寄主树种、传播媒介、松材线虫及其携带的微生物、以及环境因素等多个方面,至今其致病机理尚存争议。若要进一步深入揭示松材线虫病的致病机理,最直接、最有效的方法就是获取松材线虫致病过程中的关键基因信息,并对其进行功能分析。目前国际上已公认植物寄生线虫食道腺细胞分泌物在植物线虫与寄主互作过程早期起到关键作用,因此编码这类分泌物的基因被认为是线虫的致病相关基因。其中,在食道腺细胞特异表达的类毒液过敏原蛋白基因(Bx-vap-1)已成功在松材线虫中克隆分离,推测该基因在松材线虫寄生寄主的早期阶段发挥了重要作用,很可能是松材线虫的致病基因。本研究即以Bx-vap-1基因为研究对象,采用正向验证基因功能的方法,将Bx-vap-1基因在昆虫细胞中表达获得重组VAP蛋白,用来模拟松材线虫口腔分泌物,接种三年生马尾松,检测不同时期寄主松树防御基因的表达、萜烯含量的变化以及组织病理学变化,意从分子防御水平、生理代谢水平和组织病理学水平上的寄主响应事件来分析Bx-vap-1基因的功能,从而为松材线虫病致病机理研究提供理论基础。主要结论如下：1.利用Bac-to-Bac表达系统原理,将松材线虫Bx-vap-1基因克隆至杆状病毒转移载体pFastBacHTA,将重组质粒pFastBac-vap转化至大肠杆菌DH10Bac感受态细胞,细胞内发生转座,获得重组杆状病毒表达载体Bacmid-vap。在脂质体的介导下,转染Sf9昆虫细胞,病毒扩增3代后收获重组VAP蛋白,经SDS-PAGE和Western Blot检测验证了目的蛋白的特异性。通过对P2代杆状病毒转染细胞的M.O.I值和转染时间进行优化,最终实现了目的蛋白的大量表达。2.利用RACE技术分离克隆了马尾松α-蒎烯合成酶基因全长,并结合生物信息学软件分析了该基因编码蛋白的特性。马尾松α-蒎烯合成酶基因cDNA全长序列共1890bp,编码629个氨基酸。编码蛋白的预测分子量为71.94kDa。α-蒎烯合成酶基因编码蛋白含有1个金属结合结构域、一个天冬氨酸富集基序(DDMYD),符合单萜烯合成酶基因的典型特征。序列提交至GeneBank,获得登录号KF547035。该基因的分离克隆为研究马尾松分子防御机制提供了基因信息。3.将重组VAP蛋白作为接种体,模拟松材线虫口腔分泌物,接种三年生马尾松,接种试验表明重组VAP蛋白可以诱导马尾松α-蒎烯合成酶基因上调表达和α-蒎烯含量增加,这表明在松材线虫与松树互作过程中,线虫口腔分泌的VAP蛋白可作为激发子启动寄主的防御反应,因此编码VAP蛋白的Bx-vap-1基因是松材线虫的致病基因。4.重组VAP蛋白接种三年生马尾松,接种点附近表现出明显的感病症状,针叶干枯下垂,韧皮部坏死,木质部髓心组织褐变。细胞学观察发现VAP蛋白可以诱导马尾松细胞发生质壁分离、细胞核降解等现象,属于典型的细胞程序化死亡,说明VAP蛋白对寄主细胞有毒害作用,再次验证了Bx-vap-1基因是松材线虫的致病基因。但接种VAP蛋白没有引起马尾松的空洞化现象,空洞的形成可能还需要如细胞壁降解酶等其它致病因素的共同作用。更多还原

【Abstract】 Pine wilt disease is a fatal one caused by infection of the pine wilt nematode Bursaphelenchus xylophilus. The disease was first identified in Japan, where an outbreak occurred, and has been since spread to many countries including China, Japan, South Korea USA, Canada, Mexico and Portugal. The pathogenesis of pine wilt disease is not clear, because it is affected by multiple factors including the plant hosts, longhorned beetles, nematodes and the microbes associated with them, as well as abiotic environmental factors. Pine wilt disease has posed enormous pressure on the ecosystem and caused huge economic loss in the disease epidemic countries. Since the beginning of the1980s, pine wilt has emerged firstly and became widespread in China. It has been continuously threatening and destroying the pine ecosystem, and is one of the most dangerous biological disasters in the forest in China. To further revealed the pathogenesis of pine wilt disease, the most direct and effective way is getting the key genetic information in parasitism process, and analysising gene function.At present, it has been recognized internationally that esophageal gland cell secretion of plant parasitic nematodes play a key role in early interaction, genes encoding these secretion has been recognized as pathogenicity-related genes. Venom allergen-like protein gene (Bx-vap-1) expressed specifically in esophageal gland cells has been isolated from Bursaphelenchus xylophilus, it is inferred that Bx-vap-1gene might be pathogenic genes of Bursaphelenchus xylophilus, which plays an important role in early parasitism.In this study, Bx-vap-1gene as the research object, We take positive validation method, Bx-vap-lgene was expressed in insect cells, obtain recombinant VAP protein, to simulate esophageal gland secretion of Bursaphelenchus xylophilus, inoculate three-year old pinus massoniana. Detect pine defense genes expression, terpenes content change and histopathological changes, analyze Bx-vap-1gene function at molecular defense level, physiological metabolism level, histopathological level. Providing fundamental basis for pathogenesis research of pine wilt disease, the main conclusions are as follows:(1) The Bac to Bac baculovirus expression system was used to express Bx-vap-1gene. This expression system is efficient for producing recombinant baculovirus for expression testing in insect cells with high expression, easy screening, exact modification after transcription and no endotoxin toxicity. Fistly, Bx-vap-1gene was cloned into the pFastBacHTA vector, and the recombinant plasmid was transformed into DH10Bac competent E.coli to generate a recombinant bacmid. The recombinant bacmid DNA Was transfected into the Sf9insect cell to generate a recombinant baculovirus with cellfectin. After amplifying, the baculoviral stock was used to infect Sf9insect cells to express the recombinant VAP protein. The expressed recombinant proteins were confirmed by SDS-PAGE and Western blot analysis.(2) According to high conservative amino acids of reported a-pinene synthase gene from pinaceaes, a pair of degenerate primers were designed and partial fragment was obtained by PCR amplification. Based on this known sequence, some new primers were designed, two terminals gene sequence were cloned by3’RACE and5’ RACE respectively. After splicing the fragments of sequence, analyzed the structures and function of the coded protein by bioinformatics softwares. The full-length cDNA of a-pinene synthase gene consists of2103bp, contains a1890bp open reading frame (ORF) encoding629amino acid proteins, including N-terminal transit peptide, metal-binging domain and DDXXD motif, it has the typical characteristics of monoterpene synthase gene. The sequence has been submitted to GeneBank, accessin No. KF547035.(3) Recombinant VAP protein as inoculum to simulate esophageal gland secretion of Bursaphelenchus xylophilus, inoculate three-year old pinus massoniana. Inoculation experiment results showed:recombinant VAP protein can induce a-pinene synthetase gene up-regulated expression. This indicated that:in interaction between Bursaphelenchus xylophilus and Pinus massoniana, VAP protein secreted from Bursaphelenchus xylophilus can be regarded as elicitors to start the host defense response, hence, Bx-vap-1gene of Bursaphelenchus xylophilus which encoded VAP protein is pathogenic gene.(4) Recombinant VAP protein inoculated three-year old pinus massoniana. It appearsd obvious disease symptoms, such as, needles were wilt and droopy, phloem necrosis, xylem pith appeared brown stain. Cytological observation showd recombinant VAP protein can induce plasmolysis and nucleus degradation, this is a typical programmed cell death, this indicates that recombinant VAP protein can causes obvious damage to Pinus massoniana cell. It proved again that Bx-vap-1gene of Bursaphelenchus xylophilus is pathogenic gene. However, recombinant VAP protein didn’t cause cavitation, its formation may needs more elements combined action such as cell wall degrading enzyme.更多还原