华山松大小蠹肠道微生物群落多样性与幼虫肠道纤维素降解菌的研究毕业论文 分类号： 学校代码：XXX U D C： 研究生学号：XXX 密 级： XXX届攻读博士学位研究生学位（毕业）论文 华山松大小蠹肠道微生物群落多样性与幼虫肠道纤维 素降解菌的研究 学科专业： 野生动植物保护与利用 研究方向： 森林昆虫学 研 究 生： XXX 指导教师： XXX 教授 完成时间： XXX年4月 中国 陕西 XXX University code: XXX UDC: Postgraduate number: XXX Dissertation for Doctor Degree Northwest A GUT-ASSOCIATED MICROBIOTA DIVERSITY OF THE WHITE PINE BEETLE (DENDROCTONUS ARMANDI) AND CELLULOLYTIC MICROBIAL COMMUNITY IN ITS LARVAL GUT Major: Wildlife conservation and utilization Research field: Forestry Entomology Name of Postgraduate: Hu Xia Adviser: Prof. Chen Hui Date of submitted: April, XXX Yangling Shaanxi China 华山松大小蠹肠道微生物群落多样性与 幼虫肠道纤维素降解菌的研究 摘 要 华山松大小蠹（Dendroctonus armandi Tai and Li）作为中国秦岭巴山地区最为重要的森林害虫，严重危害森林健康和生态系统的稳定性。目前对其肠道微生物的群落结构和多样性知之甚少，本研究结合巢式PCR-DGGE、克隆测序、ARDRA、SDS-PAGE和native-PAGE技术对华山松大小蠹肠道微生物群落结构和多样性，以及幼虫肠道纤维素降解菌进行了系统的研究，主要研究结果如下： 1. 利用巢式PCR-DGGE技术分析了华山松大小蠹幼虫、蛹、雌雄成虫消化道内细菌的群落结构、多样性及其动态变化。结果表明：华山松大小蠹肠道微生物群落结构相对简单且随着发育阶段不同而发生改变，雌雄成虫的细菌群落组成差异不显著（P>0.05）；柠檬酸杆菌（Citrobacter）是华山松大小蠹幼虫、雌雄成虫肠道细菌群落的主要组分，尤其是在雌性成虫肠道细菌群落中更显优势，占细菌群落总数的80%，而蛹期甲基杆菌（Methylobacterium）占细菌群落总数的56%，为优势种群。基于Citrobacter的固氮性和Methylobacterium参与碳循环等，可以推测华山松大小蠹肠道细菌Citrobacter和Methylobacterium与华山松大小蠹的发育和营养利用密切相关。 2. 利用真菌18S rRNA区域PCR扩增与DGGE技术研究了华山松大小蠹坑道、幼虫、蛹、雌雄成虫消化道内的真菌群落。结果表明：华山松大小蠹不同发育期肠道内的真菌群落多样性指数均表现出显著差异（P<0.05）；酵母菌中的假丝酵母（Candida）是华山松大小蠹坑道、幼虫、蛹和雌雄成虫期的优势种群，尤其是在蛹期肠道真菌群落组成中更为明显，占真菌群落总数的56%，而华山松大小蠹共生蓝变真菌（长喙壳属Ophiostoma）则是华山松大小蠹坑道、幼虫、蛹和雌雄成虫期丝状真菌群落组成的最高组分。华山松大小蠹的肠道真菌大多与其消化机制、解毒机制以及信息素合成相关。 3. 通过以羧甲基纤维素钠作为唯一碳源和干果红染色法，从华山松大小蠹幼虫肠道中筛选出91株纤维素降解细菌，通过ARDRA分型为11类，对各类细菌进行形态和分子鉴定的结果显示：幼虫肠道纤维素降解菌主要是由-变形菌纲（73.6%）、-变形菌纲（8.8%）和厚壁菌门（17.6%）的细菌组成，其中-变形菌纲肠杆菌科沙雷氏菌属（Serratia）是华山松大小蠹肠道可培养细菌中最重要的纤维素降解菌，占总纤维素降解菌的49.5%，假单胞菌（Pseudomonas）占总纤维素降解菌的22%。本研究首次发现短波杆菌（Brevundimonas kwangchunensis）、泡囊短波单胞菌（B. vesicularis）、甲基杆菌（M. populi）和假黄色单胞菌（Pseudoxanthomonas mexicana）具有纤维素降解活性。 4. 通过形态和分子鉴定，证实了华山松大小蠹幼虫肠道纤维素降解真菌即为华山松大小蠹体外共生真菌秦岭细粘束孢（Leptographium qinlingensis），表明华山松大小蠹幼虫肠道的秦岭细粘束孢由成虫体外携带，经幼虫取食而进入肠道。 5. 通过硫酸铵分级沉淀和DEAE-纤维素阴离子交换柱纯化得到秦岭细粘束孢的漆酶蛋白，在SDS-PAGE和native-PAGE图谱中仅显示一个条带，为单一蛋白，分子量61 kDa，米氏常数15.3 ?M，最大反应速度372.9 U/mg。此外，1 mM和10 mM的Cl-、SO42-、NO3-对秦岭细粘束孢的漆酶活性影响不显著（P>0.05），而100 mM的 Cl-和NO3-离子对漆酶活性具有显著的抑制作用（P<0.05）。 本研究从华山松大小蠹肠道微生物的丰度、种群结构、群落功能多样性等方面探索了华山松大小蠹肠道微生物对华山松大小蠹的营养消化、发育和解毒机制的影响，研究结果丰富了华山松大小蠹肠道微生态、华山松大小蠹与微生物的内在关系，为华山松大小蠹的种群动态和纤维素的营养利用，及其综合治理提供了理论依据。 关键词：华山松大小蠹；微生物群落；巢式PCR-DGGE；纤维素酶；漆酶 GUT-ASSOCIATED MICROBIOTA DIVERSITY OF THE WHITE PINE BEETLE (DENDROCTONUS ARMANDI) AND CELLULOLYTIC MICROBIAL COMMUNITY IN ITS LARVAL GUT ABSTRACT The Chinese white pine beetle, Dendroctonus armandi Tsai and Li, is arguably the most destructive forest insect in the Qinling Mountains in Northern China. Little is known about the structure of the microbial communities associated with D. armandi, even though this wood-boring insect plays important roles in ecosystem and biological invasion processes that resulted in huge economic losses in pine forests. In this work, nested PCR-DGGE, clone sequencing, ARDRA, SDS-PAGE and native-PAGE were employed to investigate the composition and the roles of the microbial communities present in the guts of D. armandi at different developmental stages. The main points of this dissertation are listed as follows: 1. Analysis of PCR-amplified 16S rRNA gene fragments of bacteria from the guts of D. armandi larvae, pupae, as well as male and female adults revealed low complexity of bacterial communities which differed according to the developmental stage. Citrobacter spp. and Pantoea spp. predominated in larvae and adults, whereas Methylobacterium was the dominant genus at the pupal stage (56%). The main difference between the guts of male and female adults was the greater dominance of Citrobacter in females (80%). These results suggested that the bacterial community associated with D. armandi guts may influence the development of insect. 2. Analysis of PCR-amplified 18S rRNA gene fragments of fungi from the guts of D. armandi larvae, pupae, male and female adults and their galleries in Pinus armandi revealed low complexity of fungal communities which differed according to the developmental stage. Yeast of the genus Candida and filamentous fungal Ophiostoma predominated in guts and galleries of D. armandi. In particular, Candida accounted for 56% in the pupal stage, which was related to lipolysis and might support pupae physiological activities. The fungal community in D. armandi guts represented one of the factors that may interfere with insect development. 3. A total of 91 cellulolytic bacteria were isolated and assigned to 11 genotypes using amplified ribosomal DNA restriction analysis (ARDRA) in the int