共生细菌与松材线虫协同解毒寄主防御性化合物的机制研究

For its adaptation to live in pine tree, the pinewood nematode has to degrade abundant compounds produced by its host's defensive metabolism. However, studies on the genome and transcriptome of the nematode Bursaphelenchus xylophilus found that enzymes involved in degradation of terpenes are few and the metabolic pathway of α-pinene degradation is defective in the genome of the nematode. Nevertheless, detoxification enzymes in the bacteria associated with the nematode were found to be rich and the metabolic pathway of α-pinene degradation was perfect. Based on previous studies, a hypothesis is put forward as follows: during the evolutionary process, a complementary mechanism on detoxification metabolism of host's defensive compounds exists between the pinewood nematode and its symbiotic bacteria, which is in favor of the nematode's parasitism and adaptation in pine hosts. In order to verify the hypothesis, in this project, we will employ the metagenomics approaches to testify that this cooperation is common between the nematode and its symbiotic bacteria firstly. Then, we will clarify which bacteria can degrade and utilize those compounds. After that, we will define whether the detoxification metabolism of symbiotic bacteria are in favor of the nematode to adapt to hosts, by comparing the tolerances of the nematodes with or without bacteria to suffer those compounds and the differences of them in propagation on pine host. Thereby the role of symbiotic bacteria on the parasitic adaptation of the pinewood nematode to pine host will be elucidated.

松材线虫寄生在松树体内，对丰富的寄主防御性化合物的解毒代谢是松材线虫寄生适应的前提。在前期对松材线虫的基因组和转录组研究中发现，在松材线虫基因组中，缺少对松树寄主的主要化合物松萜解毒代谢的酶和代谢途径。而在其共生菌中却发现有丰富的解毒代谢酶和完整的α-松萜降解途径。根据已有研究结果，提出以下科学假说：在长期的进化过程中，松材线虫与其共生细菌可能在解毒代谢途径上形成互补，共生细菌协助松材线虫降解寄主防御性代谢产物，从而提高松材线虫对寄主的适应性。为了验证这个假说，本项目拟采用宏基因组分析的方法，首先验证共生细菌与松材线虫解毒代谢途径互补现象的普遍性；明确哪些共生细菌在主要松萜化合物的降解过程中起关键性作用。然后，通过对比实验，比较携菌和无菌松材线虫对松萜化合物的耐受性和对松树寄主的适应性，明确共生细菌的解毒代谢有利于松材线虫在松树中寄生，从而揭示共生细菌在松材线虫寄生适应性中的作用。

松材线虫是我国重要的外来入侵物种，它引起的松材线虫病导致我国松树大面积成片死亡。松材线虫体表和体内有很多附生和共生细菌，它们对松材线虫有利或不利。本项目聚焦共生细菌在松材线虫降解寄主防御性化合物松萜中的作用，主要开展了下列工作：通过GC-MS测定，明确我国主要松树树种马尾松的氯仿提取物主要成分为α-蒎烯（α-pinene）；用不同浓度的α-蒎烯处理松材线虫，发现随着α-蒎烯浓度的增加，线虫的死亡率明显增加；低浓度的α-蒎烯处理对松材线虫的繁殖没有影响，但中高浓度α-蒎烯能显著降低松材线虫线虫的繁殖量；通过以α-蒎烯作为唯一碳源的MM培养基的选择性培养，筛选出松材线虫中能降解α-蒎烯的共生菌；宏基因组及单个基因组KEGG代谢途征分析，发现这些共生菌具有完整的松萜降解代谢途径及所需酶的编码基因；通过GC-MS检测结果显示，含有共生菌的培养瓶中，α-蒎烯的量明显低于对照，且检测到代谢途征中下游的产物，表明共生细菌对α-蒎烯具有明显的降解能力；通过消毒和抗生素复合处理，发现一些优势共生菌仍很难从松材线虫体内完全清除；利用扫描电镜观察，发现消毒处理后的松材线虫体表很少有伴生细菌存在；以细菌通用引物制备FISH的荧光探针，通过荧光原位杂交和激光共聚焦显微镜观察发现，这些共生细菌主要分布在松材线虫消化道（特别是食道球附近）及生殖系统部位；为了进一步探讨这些共生细菌与松材线虫如何互作，我们构建了一个构建了真菌介导松材线虫RNAi的技术平台，以期明确松材线虫与共生细菌协同解毒的分子机制。此外，从松材线虫共生菌的分离和鉴定出一个沙雷菌（Serratia sp.），发现其培养液具有明显的杀松材线虫效果；对该菌的全基因组进行了的序列测定、组装和注释，目前正在进行包括次生代谢产物分离与鉴定、功能基因组及比较基因组分析，以期能揭示其杀线虫机制，开发作为生物防治因子的应用潜能。