黑胸散白蚁肠道内可培养的好氧与兼性厌氧共生细菌的多样性研究及共生鞭毛虫的系统发育分析
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摘要
木食性白蚁是一类能高效降解木质纤维素的昆虫,其对木质纤维素等植物源食物的消化与肠道共生微生物密切相关。据研究共生微生物在白蚁食物消化和纤维素降解方面起着重要的作用,但迄今为止,人们对共生微生物在肠道内的代谢生理与作用机制却知之甚少。据报道白蚁肠道微生物的数量在109-1011细胞之间,而且种类十分丰富,推测它们在木质纤维素的消化,利用代谢物产能,生物固氮和尿酸循环等方面可能起着重要的作用。本实验以在我国长江流域广泛分布的木食性白蚁,黑胸散白蚁(Reticulitermes chinensis Snyder)为对象,研究了该白蚁肠道共生鞭毛虫的多样性,并通过对其肠道中可培养的好氧与兼性厌氧细菌的分离、纯化和鉴定,肠道内这部分共生菌的种群结构和分布状况进行了初步分析,探讨了细菌在共生环境中可能起到的作用和对微环境产生的影响,并对其中一株细菌进行了比较详细的分类鉴定和研究。研究黑胸散白蚁肠道中可培养共生微生物的多样性,有助于了解这些微生物在复杂的代谢过程中的作用,发现和利用新的微生物资源,为阐明共生微生物与木食性白蚁之间的共生机制,和以木质纤维素为基础的生物质能源的开发奠定了理论基础。本论文的研究主要取得了以下几个方面的进展：
采用传统的平板菌落计数法,结合以16S rDNA序列为基础的系统发育学分析技术,研究了黑胸散白蚁肠道中好氧和兼性厌氧细菌的数量和种群结构。采用三种培养基从黑胸散白蚁肠道中总共分离到104株好氧和兼性厌氧菌。104株细菌根据扩增片段限制性酶切图谱分析主要分为55种ARDRA型。这55种类型的代表性菌株16S rRNA基因序列经与Genbank数据库比对分析,属于20个属。其中30.7%的细菌种类为芽孢杆菌属细菌,占绝对优势,其次是乳球菌属细菌,占20.2%,肠杆菌属细菌在三种培养基中均有发现,是分布最广泛的一类细菌。
将分离菌株的16S rRNA基因序列的和Genbank数据库中已知菌株序列进行相似性比对,表明有6株菌的基因序列与已知细菌的相似性低于97%,可能是一些新属种的细菌。本论文对其中一株细菌TM-1T进行了详细的分类学研究,该株细菌的16S rRNA基因序列与异常球菌属细菌Deinococcus gobiensis DSM 21396T的相似性最高为94%。系统发育分析表明TM-1T在系统进化树上与Deinococcus属其他种聚在一支,16S rRNA基因序列同源性约在87-94%之间。些生理生化指标及细胞壁肽聚糖成分上也有明显差异,因此将TM-1T鉴定为Deinococcus属新种。
采用实时荧光定量PCR技术还对分离细菌中的一株乳球菌属细菌Lactococcus sp. TSB-13以及肠道中的总细菌数进行了定量。以菌株Lactococcus sp.TSB-13的16S rDNA片段的特异性引物和总细菌的16S rDNA片段引物,结合实时荧光定量技术,确定在黑胸散白蚁肠道中细菌的数量约为2.06×105个/条,而乳球菌属Lactococcus sp.TSB-13在黑胸散白蚁肠道中的个数为110个/条,该菌株占整个菌群总量的0.05%。
本研究还利用分子系统学方法结合荧光原位杂交技术,首次对黑胸散白蚁肠道内共生的鞭毛虫进行了报道。发现与黑胸散白蚁共生的锐滴虫目鞭毛虫主要有3种,分别是Pyrsonympha grandis、Dinenympha exilis和D.parva,副基体纲的鞭毛虫有4种,主要属于Spirotrichonymphade、Trichonymphidae和Trichomonadidae三大类。虽然依据光学显微镜下的观察,并不是所有的鞭毛虫都得到了鉴定,但根据系统发育分析和荧光探针的原位杂交的结果,已经显示在黑胸散白蚁肠道内共生的鞭毛虫具有较高的多样性。
Wood-feeding termites are one of the most important insects that can digest lignocellulose effectively. Although it was assumed that symbiotic microbiota play important roles in the process of cellulose degradation, so far, little was known about the physiology and metabolic activities of most of the gut symbionts. It has been estimated that termites harbor a dense and diverse population of bacteria (between 109-1011 cells per gut), which may involve in the digestion of wood cellulose, nitrogen fixation and uric acid recycling. To obtain a better understanding of the microbiota inhabiting the intestinal tract of wood-feeding termites, the subterranean termite, Reticulitermes chinensis (Snyder) widely distributed in the area of Yangtze River, was used as a model. The population structure and distribution of the aerobic and facultative anaerobic bacteria in the gut of this termite was evaluated using culture-dependent method. In total 104 bacterial strains were isolated from the gut of R. chinensis with three types of media. In addition, the symbiotic flagellates were phylogenetically characterized using molecular techniques such as clonal analysis,RFLP and fluorescence in situ hybridization. All of the results form the basis for futher studies on the metabolic mechanisms of the gut microbiota, the discovery and use of new microbial resources. The main results of this study are as follows:
The quatity and community structure of the aerobic and facultative anaerobic bacteria were studied by spread plate method combined with 16S rDNA sequence-based phylogenetic analysis. In total 104 bacterial strains, which were classfied into 55 ribotypes by ARDRA patterns, were isolated from three different media. Blast search based on the 16S rRNA gene sequnces of the representatives of the 55 ribotypes showed that all of the isolates belong to 20 bacterial genera. Quatitative analysis showed that 30.7% of the isolates belong to Bacillus, representing an absolute dominant population, whereas bacteria affiliated with Lactococcus account for 20.2% in the whole community. Enterobacter bacterial isolates have been found in all three kinds of media.
Based on the 16S rRNA gene sequences of the isolates,6 newly isolated strains could be novel bacterial genus or species since their sequnces similarity are lower than 97% compared with the other bactera retrieved from Genbank. One of the isolates, Strain TM-1T exhibited maximum 16S rRNA gene sequence similarity (94%) with Deniococcus gobiensis DSM 21396T. On the basis of the phylogenetic, chemotaxonomic and phenotypic data, strain TM-1T was supposed to a novel species of the genus Deinococcus spp.
Real-time quantitative PCR method was used for measuring the abundance of bacteria and strain Lactococcus sp.TSB-13 in termite gut. PCR primers targeting the strain Lactococcus sp. TSB-13-specific region of the 16S rRNA gene were tested.The termite gut sample was analyzed by RTQ-PCR to determine the relative abundance of strain Lactococcus sp.TSB-13 16S rRNA gene compared to the total abundance of eubacterial rDNA.The rDNA fraction of strain Lactococcus sp.TSB-13 compared to all Eubacteria were 0.05%.
Phylogenetic characterization of the symbiotic flagellates in the gut of R.chinensis was also reported for the first time. With the combination of phylogentic analysis of small subunit rRNA gene sequences and fluorescence in situ hybridization techniques, three oxymonads and four parabasalids were identified. The three oxymonad protists in the intestinal gut were P. grandis, D. exilis and D. parva, whereas the identified parabasalids belonged to the order Spirotrichonymphade, Trichonymphidae and Trichomonadidae.
采用传统的平板菌落计数法,结合以16S rDNA序列为基础的系统发育学分析技术,研究了黑胸散白蚁肠道中好氧和兼性厌氧细菌的数量和种群结构。采用三种培养基从黑胸散白蚁肠道中总共分离到104株好氧和兼性厌氧菌。104株细菌根据扩增片段限制性酶切图谱分析主要分为55种ARDRA型。这55种类型的代表性菌株16S rRNA基因序列经与Genbank数据库比对分析,属于20个属。其中30.7%的细菌种类为芽孢杆菌属细菌,占绝对优势,其次是乳球菌属细菌,占20.2%,肠杆菌属细菌在三种培养基中均有发现,是分布最广泛的一类细菌。
将分离菌株的16S rRNA基因序列的和Genbank数据库中已知菌株序列进行相似性比对,表明有6株菌的基因序列与已知细菌的相似性低于97%,可能是一些新属种的细菌。本论文对其中一株细菌TM-1T进行了详细的分类学研究,该株细菌的16S rRNA基因序列与异常球菌属细菌Deinococcus gobiensis DSM 21396T的相似性最高为94%。系统发育分析表明TM-1T在系统进化树上与Deinococcus属其他种聚在一支,16S rRNA基因序列同源性约在87-94%之间。些生理生化指标及细胞壁肽聚糖成分上也有明显差异,因此将TM-1T鉴定为Deinococcus属新种。
采用实时荧光定量PCR技术还对分离细菌中的一株乳球菌属细菌Lactococcus sp. TSB-13以及肠道中的总细菌数进行了定量。以菌株Lactococcus sp.TSB-13的16S rDNA片段的特异性引物和总细菌的16S rDNA片段引物,结合实时荧光定量技术,确定在黑胸散白蚁肠道中细菌的数量约为2.06×105个/条,而乳球菌属Lactococcus sp.TSB-13在黑胸散白蚁肠道中的个数为110个/条,该菌株占整个菌群总量的0.05%。
本研究还利用分子系统学方法结合荧光原位杂交技术,首次对黑胸散白蚁肠道内共生的鞭毛虫进行了报道。发现与黑胸散白蚁共生的锐滴虫目鞭毛虫主要有3种,分别是Pyrsonympha grandis、Dinenympha exilis和D.parva,副基体纲的鞭毛虫有4种,主要属于Spirotrichonymphade、Trichonymphidae和Trichomonadidae三大类。虽然依据光学显微镜下的观察,并不是所有的鞭毛虫都得到了鉴定,但根据系统发育分析和荧光探针的原位杂交的结果,已经显示在黑胸散白蚁肠道内共生的鞭毛虫具有较高的多样性。
Wood-feeding termites are one of the most important insects that can digest lignocellulose effectively. Although it was assumed that symbiotic microbiota play important roles in the process of cellulose degradation, so far, little was known about the physiology and metabolic activities of most of the gut symbionts. It has been estimated that termites harbor a dense and diverse population of bacteria (between 109-1011 cells per gut), which may involve in the digestion of wood cellulose, nitrogen fixation and uric acid recycling. To obtain a better understanding of the microbiota inhabiting the intestinal tract of wood-feeding termites, the subterranean termite, Reticulitermes chinensis (Snyder) widely distributed in the area of Yangtze River, was used as a model. The population structure and distribution of the aerobic and facultative anaerobic bacteria in the gut of this termite was evaluated using culture-dependent method. In total 104 bacterial strains were isolated from the gut of R. chinensis with three types of media. In addition, the symbiotic flagellates were phylogenetically characterized using molecular techniques such as clonal analysis,RFLP and fluorescence in situ hybridization. All of the results form the basis for futher studies on the metabolic mechanisms of the gut microbiota, the discovery and use of new microbial resources. The main results of this study are as follows:
The quatity and community structure of the aerobic and facultative anaerobic bacteria were studied by spread plate method combined with 16S rDNA sequence-based phylogenetic analysis. In total 104 bacterial strains, which were classfied into 55 ribotypes by ARDRA patterns, were isolated from three different media. Blast search based on the 16S rRNA gene sequnces of the representatives of the 55 ribotypes showed that all of the isolates belong to 20 bacterial genera. Quatitative analysis showed that 30.7% of the isolates belong to Bacillus, representing an absolute dominant population, whereas bacteria affiliated with Lactococcus account for 20.2% in the whole community. Enterobacter bacterial isolates have been found in all three kinds of media.
Based on the 16S rRNA gene sequences of the isolates,6 newly isolated strains could be novel bacterial genus or species since their sequnces similarity are lower than 97% compared with the other bactera retrieved from Genbank. One of the isolates, Strain TM-1T exhibited maximum 16S rRNA gene sequence similarity (94%) with Deniococcus gobiensis DSM 21396T. On the basis of the phylogenetic, chemotaxonomic and phenotypic data, strain TM-1T was supposed to a novel species of the genus Deinococcus spp.
Real-time quantitative PCR method was used for measuring the abundance of bacteria and strain Lactococcus sp.TSB-13 in termite gut. PCR primers targeting the strain Lactococcus sp. TSB-13-specific region of the 16S rRNA gene were tested.The termite gut sample was analyzed by RTQ-PCR to determine the relative abundance of strain Lactococcus sp.TSB-13 16S rRNA gene compared to the total abundance of eubacterial rDNA.The rDNA fraction of strain Lactococcus sp.TSB-13 compared to all Eubacteria were 0.05%.
Phylogenetic characterization of the symbiotic flagellates in the gut of R.chinensis was also reported for the first time. With the combination of phylogentic analysis of small subunit rRNA gene sequences and fluorescence in situ hybridization techniques, three oxymonads and four parabasalids were identified. The three oxymonad protists in the intestinal gut were P. grandis, D. exilis and D. parva, whereas the identified parabasalids belonged to the order Spirotrichonymphade, Trichonymphidae and Trichomonadidae.
引文
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