【作者】 王莹；

【导师】 宋安东； 徐健；

【作者基本信息】 河南农业大学 ， 微生物学， 2015， 硕士

【摘要】 面对日益严峻的能源短缺问题与环境污染现状,寻求新型可替代的清洁能源是实现国家可持续发展的有效途径。生物质能源的探索及开发成为当今社会的热点问题。在自然界中,白蚁被公认为是最高效的木质纤维素降解反应器,并拥有对木质纤维素极高的转化利用能力。目前,一些研究者主要针对不同种类白蚁肠道菌群的结构及特点进行了研究,但对于高等白蚁肠道菌群在饮食结构多样性及饲喂过程中菌群结构的动态变化特点知之甚少。因此,在本研究中,我们采用16S rDNA焦磷酸测序技术,以我国主要分布于河南地区的高等白蚁——商城奇象白蚁(Mironasutitermes shangechengensis)为研究对象,分析不同木质纤维素含量饲喂底物(木块、玉米秸秆和滤纸)及不同饲喂时间(0、4、7和10d)对其肠道菌群多样性及群落结构组成的影响及动态变化过程。此外,本研究还将在三种木质纤维素底物下,饲喂10d后的商城奇象白蚁肠道菌群与课题组之前关于低等白蚁——扩头蔡白蚁(Tsaitermes ampliceps)饲喂15d后的肠道菌群结构相对比,研究高等白蚁与低等白蚁饲喂后肠道菌群多样性及群落结构的变化。论文的主要研究结果如下:(1)以商城奇象白蚁在不同木质纤维素含量饲喂底物及饲喂时间两个变量因素调控下提取到的27个肠道DNA样品为研究对象,通过使用454焦磷酸测序技术并结合多变量统计学原理,分析研究白蚁肠道细菌16S rDNA V1-V3区的物种多样性及其群落结构的动态变化特点。主成分分析(principal components analysis,PCA)及基于UniFrac的主坐标分析(principalcoordinate analysis,PCoA)的结果均显示,不同饲喂底物与饲喂时间下的样品组之间彼此分离,同种饲喂条件下的平行样品间存在明显的交叠现象。其中,无论是加权还是非加权的UniFrac距离分析(weighted and unweighted UniFrac distances)结果均显示,若定义木块为底物饲喂0d的样品为原生态样品,在玉米秸秆和滤纸底物下饲喂4d及7d后,白蚁肠道菌群结构与原生态样品间存在显著差异(P<0.05,Kruskal-Wallis test),然而,饲喂10d后,玉米秸秆和滤纸饲喂下的样品与原生态样品则均无显著差异(P>0.05,Kruskal-Wallis test),这表明不同饲喂底物的短期调控会引起白蚁肠道菌群结构的显著变化,随着扰动时间的延长,肠道菌群结构最终将呈现明显反弹趋势,即重新回归到原生态菌群的稳定状态。(2)在白蚁肠道菌群的动态变化过程中,我们发现共有以下5个门类的菌与反弹趋势显著相关,它们包括:螺旋体门(Spirochaetes)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、柔膜菌门(Tenericutes)以及酸杆菌门(Acidobacteria)。上述结果表明,虽然不同饲喂条件扰动下的白蚁肠道菌群结构存在一定的差异,但其中存在核心的菌群与该变化密切相关。因此,密切关注这些核心菌的变化规律,对于揭示白蚁肠道菌群反弹回归趋势的形成原因可能存在十分积极的影响作用。(3)本研究第二部分以商城奇象白蚁(高等白蚁)和扩头蔡白蚁(低等白蚁)在木块饲喂0d,玉米秸秆及滤纸分别饲喂10d和15d后提取到的21个肠道DNA样品为研究对象,通过使用454焦磷酸测序技术并结合多变量统计学原理,分析研究不同饲喂底物对两种白蚁肠道细菌16S rDNA V1-V3区的物种多样性及其群落结构的影响。主成分分析(PCA)及基于UniFrac的主坐标分析(PCoA)的结果均表明,在相同饲喂底物下,不同种类白蚁肠道菌群结构存在显著差异(P<0.05),然而,同种白蚁在不同饲喂底物下,扩头蔡白蚁的肠道菌群结构存在显著差异(P<0.05),商城奇象白蚁的肠道菌群结构则没有明显差异(P>0.05)。此外,通过对商城奇象白蚁和扩头蔡白蚁肠道菌群的丰度及组成结构的研究结果表明,两种白蚁肠道均存在以下9种优势菌群(相对丰度>1%),分别为:螺旋体门(Spirochaetes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、TG3、纤维杆菌门(Fibrobacteres)、Elusimicrobia、厚壁菌门(Firmicutes)、互养菌门(Synergistetes)和放线菌门(Acidobacteria)。上述结果表明,不同种类的白蚁,其肠道微生物的组成及丰度也差异,且玉米秸秆和滤纸饲喂底物对扩头蔡白蚁肠道菌群结构的影响大于商城奇象白蚁,这可能表明高等白蚁具有更加完善的菌群调节机制。更多还原

【Abstract】 The problem of power shortage is becoming more and more serious,looking for a new alternative and clean energy become a major challenge for the whole world.Exploration and development of biomass energy is becoming a hot issue in today’s society.In nature,Termites are considered among the most efficient bioreactors,with high caPacities for lignocellulose degradation and utilization.Recently,several studies have characterized the gut microbiota of diverse termites.However,the temporal dynamics of the gut microbiota within a given higher termite with dietary diversity are poorly understood.In our study,we employed 16S rDNA barcoded pyrosequencing analysis to investigate temporal changes in bacterial diversity and richness of the gut microbiota of wood-feeding higher termite Mironasutitermes shangchengensis under three lignocellulose content-based diets that feature wood,corn stalks,and filter paper for 0,4,7,and 10 days.In addition,we further contrast the M.shangchengensis feeding for 10 days with Reticulitermes flaviceps for 10 days both with three kinds of lignocellulosic content substrates to study the structure and diversity of higher and lower termite gut microbiota.The major resluts are as follows:First,we choose 27 DNA samples of M.shangchengensis feeding with three kinds of lignocellulose content diets for different feeding time as object,using 454-pyrosequencing analysis combine with multivariate statistical principle to investigate the dynamic changes of species diversity and community structure.The results of PCA and UniFrac-based PCoA revealed that samples of different treatment groups are separated from each other,while,parallel samples from same treatment groups exist an overlap.Weighted and unweighted UniFrac analysis showed that,compared with the group feeding with wood for 0 day,which were defined as the origin group,the groups feeding with corn stalks and filters for 4 and 7 days were both have significant changes(p<0.05,Kruskal-Wallis test).This shows that the short-term regulation of different feeding substrates may cause significant changes in the termite gut flora structure,while,the structure of intestinal flora will eventually appear an obvious rebound trend,namely,return to the steady original state.During the process of dynamic changes in the termite gut microbiota,we found 5 bacteria were significant associated with the rebound trend,including:Spirochaetes,Firmicutes,Actinobacteria,Tenericutes and Acidobacteria.The above results showed that the gut microbiota still have some core bacteria which were closely related to the changes,although there are some differences among different treatment groups.Therefore,pay more attention to these core bacteria will have positive effect on the reasons for the rebound trend.Second,we choose 21 DNA samples including M.shangchengensis feeding with wood for 0 day and feeding with corn stalks and filters for 10 days,and R.flaviceps feeding with wood for 0 day and feeding with corn stalks and filters for 15 days as study objects,using 454-pyrosequencing analysis combine with multivariate statistical principle to investigate the difference of species diversity and community structure.The results of PCA and UniFrac-based PCoA revealed that,under the same feeding substrate,there was significant difference of gut flora structure between each other(p<0.05).In the same kind of termite,during the feeding process with different substrates,there had significant difference in R.flaviceps gut microbiota(p<0.05),but no significant difference in M.shangchengensis gut microbiota(p>0.05).Besides,study of relative abundance and compositions of the M.shangchengensis and R.flaviceps gut microbiota revealed that 9 dominant bacteria(relative abundance of>1%)were found in gut microbiota,they are Spirochaetes、Bacteroidetes、Proteobacteria、TG3、Fibrobacteres、Elusimicrobia、Firmicutes、Synergistetes and Acidobacteria.Above results showed that,different kinds of termites have different composition and relative abundance of gut bacteria.The impact of different diets feeding in M.shangchengensis is greater than that in R.flaviceps bacterial community,which may indicate that higher termites may have a more perfect flora regulation mechanism.更多还原