长江中下流域草食性雁食性及肠道微生物研究

发布时间：2018-03-25 13:06

本文选题：白额雁　切入点：豆雁　出处：《中国科学技术大学》2016年博士论文

【摘要】：白额雁Anser albifrons、豆雁Anser fabalis和鸿雁Anser cygnoides是在长江中下游流域越冬的主要雁种。目前对其研究存在诸多不足。例如,需要分辨率高、精确度好的食性分析方法来补充或者替代传统的显微镜观察法,后者通常费时费力,且分辨率低。此外,这些草食性雁依赖薹草属、禾本科等草本植物提供能量。同为植食性的反刍动物等,主要依赖其肠道微生物进行消化。对于草食性雁的肠道微生物则知之甚少。针对上述薄弱环节,本论文结合分子生态学、微生物学和生物信息学等进行探究,并取得如下结果：1.安徽升金湖国家级自然保护区湿地植物基因库位于长江南岸的升金湖是重要的水鸟越冬地之一,长江流域5-10%的水鸟种群在此越冬,其丰富的植物资源为水鸟提供了充足的食物。本文以该湖泊湿地植物为研究对象,构建了草食性雁潜在食物的DNA数据库。首先通过GenBank代表性序列分析及PCR扩增检测,从8个候选基因中筛选出trnL基因作为标记基因；其后采集雁潜在食物的样本(共87份,隶属于25科,53属,70种),完整构建了升金湖冬春季节湿地植物参考基因库。该结果既为后续的湿地植物研究提供了数据基础；也为下一章使用宏条形码(metabarcoding)技术分析草食性雁的食性提供了条件。2.宏条形码(metabarcoding)研究草食性雁食性食物是影响水鸟数量与分布的重要因素,对其食物种类与组成(即食性)进行分析即成为水鸟生态学研究的重点之一。本研究引入宏条形码(metabarcoding)技术对白额雁和豆雁进行食性分析。结果发现,白额雁食物中73%的序列为禾本科；豆雁食物中99%的序列为薹草属；大部分食物在物种水平得到鉴定。同步的显微镜观察发现,白额雁的主要食物也是禾本科；而豆雁食物中另外发现约13%的禾本科：有两种食物仅在显微镜观察中发现。两种方法得到的主要食物类型一致,宏条形码得到的信息更多,分辨率更高。结果的差异可能源于PCR偏好性扩增等。两种雁食谱的差异与分化可能与其生态学特征等相关。本研究认为,宏条形码技术值得被引入水鸟食性的分析,可以加速样品处理的进度,并提供更高分辨率、更客观的结果；同时,显微镜观察也值得保留,以提供准确的定量结果。3.草食性雁肠道微生物与食性相关本文研究的三种雁主要依赖薹草属、禾本科等草本植物提供能量。然而,它们的肠道微生物情况如何,是否与食性相关等,则缺乏研究。本研究发现,雁肠道微生物主要由厚壁菌Firmicutes(49.70%)和变形菌Proteobacteria(23.80%)组成。然而,不同物种、不同食性的雁,其肠道微生物多样性和群落结构存在显著性差异。共同栖息于鄱阳湖的三个物种,豆雁肠道微生物多样性显著高于白额雁和鸿雁,并且富集最高丰度的变形菌(50.26%)；鸿雁肠道微生物多样性最低,其中厚壁菌(78.99%)最多。升金湖白额雁的多样性高于鄱阳湖白额雁,并且有大量放线菌Actinobacteria(26.68%)。镜检发现鄱阳湖三种雁均取食薹草属；而升金湖白额雁则同时取食薹草属和禾本科。Mantel检验证实升金湖白额雁肠道微生物的遗传结构与食物组成(薹草属与禾本科的比例)显著相关。可见,遗传和食物都会影响雁肠道微生物的结构。雁肠道微生物基本组成及影响因素研究,为揭示肠道微生物在水鸟迁徙与适应过程中的作用提供了重要参考。4.草食性雁肠道微生物互作与功能微生物之间的相互作用不仅是微生物多样性的重要组成部分,同时也会影响其群落功能的执行。基于16S数据,本研究引入RMT(random matrix theory,随机矩阵理论)模型,构建雁肠道微生物分子生态网络(molecular ecology networks)。结果表明,升金湖与鄱阳湖的白额雁之间、鄱阳湖三种雁之间,在分子生态网络的结构、节点特征等方面存在明显差异：证实肠道微生物之间的互作具有较高的多样性。同时,我们引入PICRUSt(phylogenetic investigation of communities by reconstruction of unobserved states)对微生物功能进行预测。发现,所有样品组的微生物都执行一些保守的功能,主要是参与物质和能量代谢的基因。这些研究主要基于16S测序数据进行推测。微生物之间真正的相互作用以及功能发挥,仍需要功能基因组学等技术进行直接解析。上述研究,1)首次构建了安徽升金湖国家级自然保护区冬春季节湿地植物基因库；2)引入了宏条形码技术分析草食性雁的食性,并证实其可用性；3)首次综合阐述多种雁肠道微生物组成与结构；4)引入分子生态网络和PICRUSt方法解析雁肠道微生物的互作与功能。本研究表明,交叉学科的应用可以实现对雁等鸟类的深入认识和理解。本论文的主要创新点为：1)在方法学上,对传统的食性分析方法进行改善；2)在研究内容上,阐述了多种雁的雁肠道微生物结构与功能,并解析了其与食物的相关性。
[Abstract]:White fronted goose Anser albifrons, Anser fabalis and Anser cygnoides in bean goose goose is the main species in winter in the Yangtze River Basin. The study has many deficiencies. For example, need high resolution and good accuracy of the diet analysis method to supplement or replace the traditional microscope observation method, which is usually time-consuming, and low resolution. In addition, these wild herbivorous genus of Gramineae on bolting, herbs provide energy. Same as herbivorous ruminant animal, mainly depends on its intestinal microbial digestion. The intestinal microflora of herbivorous goose is poorly understood. In view of the weak link, this paper combines molecular ecology, microbiology and bioinformatics research results are as follows: located in the South Bank of the Yangtze River wetland plant gene pool of Anhui 1. liters of Jinhu National Nature Reserve Jinhu is an important water bird more The winter of the Yangtze River Basin 5-10% and wintering waterfowl population, its rich plant resources to provide adequate food for waterfowl. In this paper, the lake wetland plant as the research object, construct the herbivorous wild goose potential food DNA database. Firstly, GenBank representative sequence analysis and PCR analysis, from the 8 candidate the gene trnL gene was selected as marker genes; followed by the acquisition of potential food goose samples (87 samples, belonging to 25 families, 53 genera, 70 species), build a complete rise in spring and winter wetland plants in Jinhu reference gene pool. This result not only for further study of wetland plant data to provide a basis for; the next chapter use the macro code (metabarcoding) provides the conditions for the.2. macro barcoding diet of herbivorous wild goose (metabarcoding) of herbivorous geese feeding food is influence the number and distribution of waterfowl important factors for their food Species and composition (instant) analysis is to become one of the key research. The introduction of waterfowl Ecology (metabarcoding) technology on the macro code amount of geese and bean for food analysis. The results showed that the sequence of white fronted goose food in 73% of Gramineae; food for 99% series bean sprouts. Most of the food is obtained; the identification at the species level. Observed synchronous microscope, the main food is the white fronted goose grass; and bean food also found that about 13% of the Gramineae: there are two kinds of foods found only in microscopy. The main types of food have been two methods, more information from macro bar code, higher resolution. The differences may be due to the preference of PCR amplification. The difference between the two wild goose recipes and differentiation may be related to ecological characteristics. This study suggests that macro barcode technology is worth cited Analysis of waterfowl feeding habits, can accelerate the progress of sample processing, and provide higher resolution, more objective results; at the same time, microscope observation also worth preserving, with three kinds of wild geese provide accurate quantitative results.3. herbivorous gut microbes and feeding the geese related the main research on bolting of gramineous herbs. How to provide energy. However, their gut microbes, and feeding habits, is lack of research. The study found that the goose gut microbes mainly composed of Firmicutes and proteobacteria Firmicutes (49.70%) Proteobacteria (23.80%). However, different species, different feeding habits of the wild goose, there was a significant difference between its intestinal microflora diversity and community structure. Three species inhabit in Poyang Lake, intestinal microbial diversity in bean was significantly higher than that of white fronted goose and swan goose, and the highest abundance of Proteobacteria enrichment (50.26%); Goose intestinal microbial diversity is lowest, the Firmicutes (78.99% liters). Most diversity in Jinhu is higher than that of Poyang Lake white fronted goose white fronted goose, and there are a large number of actinomycetes Actinobacteria (26.68%). Microscopic examination showed that three Poyang Lake geese are feeding Tai Humulus; genetic structure and food or Jinhu white fronted goose while feeding sprouts genus of Gramineae and.Mantel examination confirmed the Jinhu white fronted goose intestinal microbial composition (Carex and Gramineae ratio) were significantly correlated. Therefore, genetic and food will affect the structure of wild goose gut microbes. Factors of goose intestinal microbial basic composition and influence, in order to reveal the intestinal microflora in migratory waterbirds and adapt to the process of the provides an important reference for.4. herbivorous wild goose intestinal microbial interactions and interactions between microorganisms is not only an important part of microbial diversity, but also affect The community function implementation. Based on the 16S data, this study adopts RMT (random matrix theory, random matrix theory) model, construct the goose intestinal microbial molecular ecology network (molecular ecology networks). The results show that between Jinhu and Poyang Lake rose three between Poyang Lake white fronted goose, goose, structure in molecular ecology network, there are significant differences between the node characteristics: confirmed the interaction between intestinal microorganisms with high diversity. At the same time, we introduce PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved States) to predict the microbial function. All samples found that microorganisms perform some conserved function is mainly involved in material and energy metabolism these studies mainly 16S gene. The sequencing data were estimated. Based on the interaction between real and microbial function, Functional genomics technology still need direct analysis. The above research, 1) was constructed for the first time in Anhui Jinhu National Nature Protection Area in winter and spring season wetland plant gene pool; 2) introduced the feeding habits of herbivorous wild macro barcode technology, and confirm its availability; 3) for the first time a comprehensive set of a variety of wild goose intestinal microbial composition with the structure; 4) interaction into the molecular ecology network and the analytic method of PICRUSt goose intestinal microbial and function. The research shows that the application of cross discipline can achieve in-depth knowledge and understanding of the wild birds. The main innovations are as follows: 1) in the methodology, the analysis method of the traditional diet to improve; 2) in the research content, elaborated a variety of wild goose goose intestinal microbial structure and function, and analyzes its relationship with food.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q958

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