【作者】 张锴；

【导师】 张星耀；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2010， 博士

【摘要】 随着全球一体化和国际社会交往与贸易往来的与日俱增,外来有害生物随人口流动、动植物及其产品跨国扩散日益严重,给入侵地生态系统和国土安全造成巨大的灾害性损失。松材线虫(Bursaphelenchu xylophilus(Steiner&Buhrer)Nickle)已经成为世界上重要的检疫对象和我国有史以来危害最为严重的林业外来有害生物。作为一种外来入侵物种,该病发现之日起就对中国的松林生态系统造成了巨大的破坏和冲击。基于经典与现代生物学的理论与方法,国内外众多学者对松材线虫的生物学、生态学和致病性等进行了深入研究,并在病害控制研究上取得了积极进展,但在实际生产应用过程中,快捷、简便而又行之有效的控制措施尚有待研究和推广。松材线虫病是一个受寄主松树、松材线虫、环境条件、树体内微生物以及媒介昆虫等多方面因素影响的复杂体系,其致病过程也是一个复杂的、受多因素影响的过程。本研究基于前人对松材线虫致病过程的认识,以我国南方乡土树种马尾松为研究对象,利用采自浙江省松材线虫病疫区内马尾松样品中分离得到松材线虫虫株接种供试马尾松苗木,并应用分子抑制性消减杂交技术(SSH)技术,以松材线虫接种24h、72h的马尾松苗为实验方(tester),以接种无菌水为对照的健康马尾松苗为驱动方(driver)构建抗松材线虫差异表达的抑制消减cDNA文库,研究分析了接种前后寄主马尾松苗基因表达的差异。通过BLAST对比分析,获得了一批涉及胁迫响应、信号传递及能量代谢等功能的差异基因。本文的研究结果为揭示松树感染松材线虫后的相关病理学响应的分子机制、论证松材线虫病的致病机理,从而达到对病害的可持续控制都提供了科学依据。1.通过预备试验对接种用松材线虫株系进行了有效的筛选。采用真菌平板培养法研究了不同松材线虫株系的繁殖能力。实验结果表明,所有的4个线虫株系都在灰葡萄孢平板上表现出了良好的生长繁殖情况。此外,供试的4个松材线虫株系在繁殖结构方面具有明显差异。平板培养10d后,NXY03株系的繁殖倍数最高,达到357倍,而最低的NXY14株系的繁殖倍数仅为193倍;在性比方面,4种松材线虫株系表现出类似的繁殖趋势,平均值为3.34左右;幼虫/成虫比方面,NXY03株系的幼虫/成虫比最高,达到了3.3,而NXY01株系最低,只有1.9。通过人工接种方法,比较了供试松材线虫株系在黑松枝段内的迁移扩散和繁殖能力。结果表明,松材线虫NXY03在黑松枝段内迁移能力更强,表现在以更快的速度和更大的种群数量穿过黑松枝段。通过试验。在繁殖倍数方面,接种20d后,松材线虫NXY03株系的繁殖倍数明显高于其它线虫株系,达到了16倍,最终繁殖线虫数量约为3200.0±100.0条,与其它线虫株系具有显著差异。通过比较发现,接种四个株系的松材线虫的马尾松苗在症状最早出现时间、发病与死亡速度,感病指数几个指标间存在显著差异,经过分析,NXY03株系的感病能力最强。通过预备试验证明,供试的四个线虫株系在繁殖能力、迁移扩散能力以及致病性几个方面均具有明显差异,其中松材线虫舟山株系(NXY03)在种群活力、繁殖能力、迁移扩散能力和致病能力方面均明显高于其他3个株系,综合试验分析结果,选取该株系线虫作为后期接种的供试松材线虫株系。2.通过试验,构建了马尾松抗松材线虫抑制消减文库,并对接种前后供试马尾松苗基因表达的差异进行了分析。提取样品总RNA后,通过SMART质检、Rsa I酶切、接头验证、差减杂交、PCR验证等一系列操作,分别成功构建了两组差减文库。斑点杂交后通过信号扫描分析共计得到665个阳性克隆,筛选出150个阳性菌落进行测序,得到144个克隆的有效序列。测序结果在NCBI数据库中经Blast比对共得到141个有效比对结果。其中,27个和已知功能的蛋白有高度的相似性;3个保守待测蛋白;111个为未知功能蛋白和假定蛋白。功能已知的蛋白基因分别来自毛果杨、蓖麻、拟南芥、玉米等物种,功能涉及到:参与植物抗病反应、编码胁迫耐受蛋白、参与结构和功能代谢合成途径、参与信号转导和调节途径以及能量代谢等。在本试验构建的SSH文库中,有与细胞分裂生长相关的60s核糖体蛋白L38;参与植物防卫反应的查尔酮合成酶、钙结合蛋白等;参与信号转导和调节途径的NPG1、组氨酸激酶受体等;胁迫耐受蛋白,如热激蛋白(HSP)、金属硫蛋白(MT3)、核糖体蛋白L15等;参与结构和功能代谢合成途径的核糖体框架蛋白(EMB1080)、ATP结合蛋白;参与次生代谢途径的肉桂酸脱氢酶(CAD)等。接种具有较强毒力的松材线虫后的很短时间内,当激发子与受体识别后,引起树体内部细胞质Ca2+浓度的变化。Ca2+离子流进入细胞,并产生离子通道,从而发出钙信号。而活性氧随后骤增,由活性氧激活细胞质转录因子,并激活防卫反应基因。之后,胁迫表达基因、抗病基因的表达也随之发生变化,随着病程的加深和寄主自身防卫反应机制的激活,其表达量也逐渐上升。而糖类代谢响应基因的差异表达证明,在接种松材线虫后,供试马尾松苗寄主还会通过加强呼吸作用的糖酵解过程以增强细胞的抗逆性。以上试验结果显示,接种松材线虫强毒株系后,寄主马尾松的基因表达在DNA的转录和翻译水平上的调控、抗性基因的参与、防御激活的次生代谢途径、受体激酶等信号分子的调控作用等方面均产生了不同程度的表达差异。这些差异的出现,可能与松材线虫侵染后寄主与病原互作的分子机制相关联。但是这些差异表达的基因是否真正具有抗病相关功能还需进一步的研究。总而言之,通过试验证明了供试马尾松接种松材线虫后,相关的抗逆、抗病基因在病程中得到上调表达,并参与了防卫反应相关信号的传递及能量代谢等机制的调控和表达。相关的研究结果对进一步探索松材线虫与寄主马尾松互作的分子响应机制,阐明该病害的致病机理具有重要意义!更多还原

【Abstract】 With the development of the global integration and international exchanging and trading, the invasive alien species dispersal causes a large damage to the ecology system of the country invaded. Pine wood nematode (Bursaphelenchus xylophilus), as an agent of pine wilt disease and the important quarantine target of many countries around the world, has been the most serious invasive pest in China. As an invasive alien species, this disease caused great destruction and impact to pine forest ecosystems since it had been found in China. Based on the classic and modern biology theory and method, many scientists focused on the biology, ecology and pathogenicity and so on from all over the world, but until recently there were no effective controlling measures to the pine wilt disease.The pine wood nematode disease is a complex system which would be influenced both by nematodes, host pine, environmental conditions, associated fungi, vector insects and other factors. The pathogenic process of this disease can also be influenced by a number of factors. Based on the prophase understanding of pine wilt disease, the genetic expression difference of native tree species Pinus massoniana seedlings before and after artificial inoculation had been analyzed with the method of molecular suppression subtractive hybridization in this study. A suppression subtractive hybridization (SSH) cDNA library was constructed with Pinus massoniana induced by Bursaphelenchus xylophilus. The branch was induced with 24 and 72 hour as tester, the non-induced branch as driver. The pine wood nematode strains for vaccination were isolated from Zhejiang Province in the South of China. The BLAST homology search revealed that these genes played varies functions in response to stress, signal transduction, energy metabolism and transportation etc. The results of the study could provide the scientific basis for demonstrating the pathology response mechanism of the disease as well as the molecular mechanism of pathogenicity. In addition, the results of this study could also give science data to provide an academic support for the sustainable management of pine wilt disease accordingly.Through preliminary tests, the results are as follows.Firstly, the population propagation ability of different B.xylophilus strains was analyzed. The population propagation ability of four B.xylophilus species was analyzed with culture method. The results of the test showed that both four B.xylophilus species have high propagation rate, suitable sex ratio and larvae/adult proportion on the Botrytis cinerea culture. In addition, four B.xylophilus strains have significantly different in propagating structure. B. xylophilus strain NXY03 exhibited the potential high propagating ability with ten days growth on the Botrytis cinerea culture. The propagation multiples of NXY03 reached a maximum of 357 times while the strain NXY14 is only 193 times as the lowest. Four species of pine wood nematode strains showed a similar trend of propagation in sex ratio mean 3.34. At the end, NXY03 lines a maximum of 3.3 on larvae/adult proportion, and NXY01 lines the minimum of 1.9.Secondly, the dispersal and propagation ability of different B.xylophilus species were compared.The dispersal and propagation ability of four B.xylophilus strains in Pinus thunbergii shoot section were analyzed with artificial inoculation. Compared with other strains, NXY03 could pass through the shoot section of P.thunbergii with a higher speed and less time. The reproduction multiple of NXY03 is 16 which was significantly higher than that of other strains in the section for 20 days. The final number of NXY03 strain was obviously different with other three strains as 3200±100.Thirdly, the virulence of different strains was compared. By comparison, the time of symptom appeared, infection rate and decease infection index of Pinus massoniana seedlings with the inoculation of four strains were all showed significantly different. NXY03 strain has the strongest virulence after analysis.The pilot experiment proved that the four nematode strains had significantly different in the reproduction ability, dispersal ability and virulence. The strain of NXY03 that isolated from Zhoushan city showed the highest ability in the population dynamics, proliferation ability, dispersal ability and pathogenic capabilities. With the analysis of the test result the author select the strain of NXY03 as the test sample in the latter part of the experiment.Tow suppression subtractive hybridization (SSH) cDNA libraries were constructed to Pinus massoniana seedlings that were induced with NXY03 selected from preliminary tests.Through testing, the genetic expression difference of Pinus massoniana seedlings before and after artificial inoculation had been analyzed. SSH libraries were successfully built with SMART method after the total RNA samples were extracted. A total of 665 positive clones were obtained with dot blot. And then 150 positive colons were filtered out for sequencing test which finally got 144 availed sequences. After the Sequencing Blast in the NCBI database, 141 effective results had been showed. Of these, 27 sequences have a high degree of similarity to the proteins that the function has been known which mostly got from Ricinus communi, Populus trichocarpa, Arabidopsis thaliana and Zea mays. There of them were conservative hypothetical proteins, and 111 of these were functional unknown or predicted protein.Features of functional known protein involves in: participating the disease resistance of plants, coding stress tolerance proteins, participation in structure and function metabolism, as well as participating signal transduction and energy transportation etc.In the SSH library that constructed in the experiment, many differently expressed gene were found. These genes involved cell division grows (60s ribosome protein L38), plant defense response (Chalcone synthase, Calcium-binding protein), signal transduction and adjustment (NPG1), stress tolerance (HSP, MT3, ribosome protein L15), structure and function metabolism (EMB1080, CAD) and so on.After artificial inoculation, the exciton recognized acceptor in very short time. The recognition response caused density changes of Ca2+ in the host cytoplasm. Then the Ca2+ stream enters the cell, and produces the ion channel, thus the calcium signal was sending out. The active oxygen suddenly increases, and activated cytoplasm transcription factor and the defense response gene. Afterward, the coercion expresses the gene, the disease-resistant gene expression also to change along with it. The host defense response mechanism was activated with the course of the disease, the expression quantity of the defense response gene also gradually rises in the same time. In addition, the difference expression of carbohydrate metabolism response gene showed that the host pine tree strengthen the respiration and sugar zymolysis process to strengthen cell’s resistance after artificial inoculation.The above test result improved that, the host pine’s gene expression had the varying degree expression difference on DNA duplication and translation, resistant gene participation, secondary metabolism control, signaling molecule control after artificial inoculation. The appearance of the difference expression result is likely associated with the interaction molecular mechanism between the host and the pathogen pine wood nematode. But whether the function of these difference expression genes are related to the disease-resistant also needs further research.The analysis results of secquences blast proved that the expression of disease resistance genes are up regulated in the course of disease after inoculated with pine wood nematodes. These genes all participate in the regulation and expression of the defense reaction-related signal transduction and energy metabolism mechanisms. The research results also provide the basic datas which are relevant to further study of the molecular interaction mechanism between pine wood nematode and the host pine trees, and could play an important role in clarifying the pathogenic mechanism of the disease.更多还原