【作者】 杨红；

【导师】 洪华珠； Andreas Brune；

【作者基本信息】 华中师范大学 ， 农药学， 2004， 博士

【摘要】 白蚁是国际生态研究中的五大害虫研究对象之一。由于其种类繁多，食性广泛，因此对人类生产和生活所造成的危害是巨大的。全世界每年因白蚁危害造成的经济损失数以亿计。已有的研究表明，低等木食性白蚁对木质纤维素等植物源食物的消化主要与其肠道共生微生物有关。但目前为止，人们对其肠道共生微生物在食物消化，代谢生理等方面的作用却知之甚少。为了对木食性白蚁肠道共生微生物有一个全面的了解，以揭示白蚁肠道共生微生物在木质纤维素消化中的作用，本论文在国际上首次报道了应用分子克隆，DNA指纹分析，及荧光原位杂交等多种先进的分子生物学技术，以散白蚁Reticulitermes santonensis(Feytaud)为模式系统研究低等木食性白蚁肠道内不同微生态环境中细菌的区系结构及多样化和主要细菌类群在该白蚁肠道的分布与定位等问题，并研究了与散白蚁Reticulitermes flavipes(Kollar)肠道共生鞭毛虫P．vertens相关的共生细菌的系统发育类型。为进一步阐明这些肠道共生微生物在复杂的代谢过程中的作用，并在此基础上探索通过消灭肠道共生微生物使白蚁由于“缺共生生物病”而死亡的途径奠定了理论基础。对采取科学措施进行木食性白蚁的综合防治具有重大的实际意义。本研究的主要结果如下： 1．在国际上首次研究了与木食性散白蚁Reticulitermes flavipes(Kollar)肠道共生鞭毛虫Pyrsonympha vertens相关的共生细菌的系统发育类型。借助显微操作仪从该白蚁肠液中分离出单一鞭毛虫P．vertens，运用分子克隆实验技术构建了与散白蚁R．flavipes肠道内共生鞭毛虫P．vertens相关的共生细菌16S rRNA基因文库，通过对挑选克隆的RFLP分析选择了5个克隆进行全序列测定，其中4个克隆属于“白蚁菌群1”(Termite group 1)，另1个克隆属于滑行菌门(Cytophaga-Flexibacter-Bacteroides phylum,CFB phylum)的拟杆菌。根据测序克隆的RFLP图谱所代表的克隆数，说明“白蚁菌群1”和CFB phylum的拟杆菌均为与鞭毛虫P．vertens相关的优势共生细菌类群。 2．本研究以生活于地下的散白蚁Reticulitermes santonensis(Feytaud)为对象，在国内、外通过分子克隆和系统发育分析的方法首次报道了低等木食性白蚁消化道中四个不同的微生态环境，即中肠，后肠肠壁，后肠肠液，和肠道共生鞭毛虫中细菌的区系结构。结果表明，这些不同微环境中细菌的主要类群为革兰氏阳性菌(主要为梭状芽孢杆菌，链球菌和支原体相关克隆)，滑行菌门的细菌，螺旋体和一些紫细菌。这些类群的细菌不均匀地分布蜘博士学位论文DOCI’01挤L DISSERTArflON于肠道四个不同的微生态环境中。在该白蚁肠道内最大的一个细菌类群为所谓的“白蚁菌群1”门的细菌，迄今尚无纯培养的分离株。绝大多数“白蚁菌群1”门的克隆与鞭毛虫相关，并在系统发育树上形成两个不同的簇，该簇的克隆仅从散白蚁忍santonensz’s及其它相近的价tjcu了z’t~es的肠道中得到。其它类群的细菌克隆在系统发育树上也常常形成独立的分支，与它们最邻近的克隆也大多来自其它相近的白蚁种类的肠道。说明白蚁肠道微生物与其宿主的共进化关系。本研究表明白蚁肠道微生物区系的多样性反映了白蚁肠道微生态环境的异质性。3.以165 rDNA为标记基因用末端限制性片段长度多态性分析(terminal restrietionfr铭ment length Polymorphism，T-RFLp)技术研究了散白蚁鹿tjeu11’termes santonensjs肠道不同 微环境细菌区系结构的多样性。用五力岁I消化细菌165 rRNA基因，进行T-RFLP指纹图谱 分析发现散自蚁尤朋刀tonensz’s(Feytaud)肠道四个不同部分细菌区系结构的多样性差别 很大。后肠肠液及肠壁相关细菌区系的多样性(12~巧个峰)明显高于中肠和鞭毛虫相关 细菌的多样性(3一5个峰)。综合人勿，I消化，八勿，卜Tail消化及Tasl酶消化后的不盯LP 指纹图谱分析结果可见，在中肠基因文库中，最主要的细菌类群为链球菌和梭状芽抱杆菌: 鞭毛虫相关细菌中的优势类群依次为“白蚁菌群1”，螺旋体，和梭状芽抱杆菌:后肠肠液 中的优势细菌类群也为“白蚁菌群1”，螺旋体，和梭状芽抱杆菌，此外还有链球菌，支原 体，及紫细菌等;后肠肠壁相关细菌优势类群为“白蚁菌群1”，梭状芽抱杆菌，和CFB phylum等。4.采J一月多种细菌种群特异性探针本论文以荧光原位杂交技术对低等木食性白蚁，散白蚁尺 ssntonensl’s肠道共生细菌在肠道不同微环境的分布及定位进行了研究。结果表明，不同细 菌类群在散白蚁左santonensz’s肠道的分布是不同的。在该白蚁肠道最占优势的“白蚁菌 群1”类细菌主要是巧尹‘口尹粉阴夕ha verten:和Trichonympha agilis这两种鞭毛虫的内共生菌， 随着两种鞭毛虫的分布而遍布于肠腔各处及靠肠壁的区域。CFB phylum的细菌主要分布于 肠液和肠壁，少数与鞭毛虫相关。低G+C含量的革兰氏阳性菌部分分布于中肠和后肠的肠壁， 也有部分分布于肠液中。更多还原

【Abstract】 Termites are one of the five most important pests studied by the international research of ecology. Owing to the numerous species and widespread food resources, termites cause a tremendous amount of economic damage to the society. Billions of dollars are lost in the world every year because of termites.It has been shown that the symbiotic gut microbiota in wood-feeding lower termites plays an important role in the digestion of different plant components. However, till recently, very little was known about how the gut microbiota is involved in digestion.To obtain a better understanding of the microbiota inhabiting the intestinal tract of wood-feeding lower termites, the European subterranean termite, Reticulitermes santonensis (Feytaud) (Isoptera: Rhinotermitidae), was used as a model system. The diversity and structure of the bacterial community in the different niches of the gut, as well as the distribution and location of major bacterial phylotypes within the intestinal tract was evaluated using various molecular techniques such as clonal analysis, DNA fingerprinting, and fluorescence in situ hybridization. In addition, the symbiotic bacteria associated with the flagellate, Pyrsonympha vertens in the gut of the closely related Eastern subterranean termite, Reticulitermes flavipes (Kollar), were phylogenetically characterized. AH of the results were reported for the first time in the world, which form the basis for further studies on the metabolic mechanisms of the gut microbiota, and for the new strategies to control wood-feeding lower termites by moving out the symbiotic microorganisms. The main results of this study are as follows:1. This was the first report on the phylogenetic characterization of the symbiotic bacteria associated with the symbiotic flagellate Pyrsonympha vertens in the gut of Reticulitermes flavipes. Single cells of P. vertens were isolated from diluted gut fluid of this termite with the aid of a micromanipulator. A clone library of the 16S rRNA genes of the bacteria associated with P. vertens was established. Clones were sorted based on the restriction fragment length polymorphism (RFLP) analysis, and the almost complete 16S rRNA genes of five clones were sequenced. Four of these clones were affiliated with the so-called Termite group 1 (TG-1) phylum, and one clone was affiliated with the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. The ratio of the clone frequencies of the two phylotypes revealed that bacteria affiliated with these two phyla were dominant in P. vertens.2. The bacterial microbiota in the four major intestinal habitats of Reticulitermes santonensis - the midgut, the wall of the hindgut paunch, the hindgut fluid, and the intestinal protozoa - were characterized by cloning and sequence analysis and by molecular fingerprinting (terminal restriction fragment length polymorphism; T-RFLP). The community comprised representatives of several phyla, including gram-positive bacteria (mainly clostridia, streptococci, and Mycoplasmatales-related clones), members of the CFB phylum, spirochetes, and a number of proteobacteria, all of which were unevenly distributed among the four fractions. The largest group of clones fell into the so-called TG-1 phylum, which has no cultivated representatives. The majorityof the TG-1 clones was associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most-closely related termites, supporting the concept of co-evolution between gut microbiota and host. This study provides conclusive evidence that the diversity of the gut microbial community reflects niche heterogeneity in更多还原