本文選題：白額雁　切入點(diǎn)：豆雁　出處：《中國科學(xué)技術(shù)大學(xué)》2016年博士論文

【摘要】：白額雁Anser albifrons、豆雁Anser fabalis和鴻雁Anser cygnoides是在長江中下游流域越冬的主要雁種。目前對其研究存在諸多不足。例如,需要分辨率高、精確度好的食性分析方法來補(bǔ)充或者替代傳統(tǒng)的顯微鏡觀察法,后者通常費(fèi)時(shí)費(fèi)力,且分辨率低。此外,這些草食性雁依賴薹草屬、禾本科等草本植物提供能量。同為植食性的反芻動(dòng)物等,主要依賴其腸道微生物進(jìn)行消化。對于草食性雁的腸道微生物則知之甚少。針對上述薄弱環(huán)節(jié),本論文結(jié)合分子生態(tài)學(xué)、微生物學(xué)和生物信息學(xué)等進(jìn)行探究,并取得如下結(jié)果：1.安徽升金湖國家級(jí)自然保護(hù)區(qū)濕地植物基因庫位于長江南岸的升金湖是重要的水鳥越冬地之一,長江流域5-10%的水鳥種群在此越冬,其豐富的植物資源為水鳥提供了充足的食物。本文以該湖泊濕地植物為研究對象,構(gòu)建了草食性雁潛在食物的DNA數(shù)據(jù)庫。首先通過GenBank代表性序列分析及PCR擴(kuò)增檢測,從8個(gè)候選基因中篩選出trnL基因作為標(biāo)記基因；其后采集雁潛在食物的樣本(共87份,隸屬于25科,53屬,70種),完整構(gòu)建了升金湖冬春季節(jié)濕地植物參考基因庫。該結(jié)果既為后續(xù)的濕地植物研究提供了數(shù)據(jù)基礎(chǔ)；也為下一章使用宏條形碼(metabarcoding)技術(shù)分析草食性雁的食性提供了條件。2.宏條形碼(metabarcoding)研究草食性雁食性食物是影響水鳥數(shù)量與分布的重要因素,對其食物種類與組成(即食性)進(jìn)行分析即成為水鳥生態(tài)學(xué)研究的重點(diǎn)之一。本研究引入宏條形碼(metabarcoding)技術(shù)對白額雁和豆雁進(jìn)行食性分析。結(jié)果發(fā)現(xiàn),白額雁食物中73%的序列為禾本科；豆雁食物中99%的序列為薹草屬；大部分食物在物種水平得到鑒定。同步的顯微鏡觀察發(fā)現(xiàn),白額雁的主要食物也是禾本科；而豆雁食物中另外發(fā)現(xiàn)約13%的禾本科：有兩種食物僅在顯微鏡觀察中發(fā)現(xiàn)。兩種方法得到的主要食物類型一致,宏條形碼得到的信息更多,分辨率更高。結(jié)果的差異可能源于PCR偏好性擴(kuò)增等。兩種雁食譜的差異與分化可能與其生態(tài)學(xué)特征等相關(guān)。本研究認(rèn)為,宏條形碼技術(shù)值得被引入水鳥食性的分析,可以加速樣品處理的進(jìn)度,并提供更高分辨率、更客觀的結(jié)果；同時(shí),顯微鏡觀察也值得保留,以提供準(zhǔn)確的定量結(jié)果。3.草食性雁腸道微生物與食性相關(guān)本文研究的三種雁主要依賴薹草屬、禾本科等草本植物提供能量。然而,它們的腸道微生物情況如何,是否與食性相關(guān)等,則缺乏研究。本研究發(fā)現(xiàn),雁腸道微生物主要由厚壁菌Firmicutes(49.70%)和變形菌Proteobacteria(23.80%)組成。然而,不同物種、不同食性的雁,其腸道微生物多樣性和群落結(jié)構(gòu)存在顯著性差異。共同棲息于鄱陽湖的三個(gè)物種,豆雁腸道微生物多樣性顯著高于白額雁和鴻雁,并且富集最高豐度的變形菌(50.26%)；鴻雁腸道微生物多樣性最低,其中厚壁菌(78.99%)最多。升金湖白額雁的多樣性高于鄱陽湖白額雁,并且有大量放線菌Actinobacteria(26.68%)。鏡檢發(fā)現(xiàn)鄱陽湖三種雁均取食薹草屬；而升金湖白額雁則同時(shí)取食薹草屬和禾本科。Mantel檢驗(yàn)證實(shí)升金湖白額雁腸道微生物的遺傳結(jié)構(gòu)與食物組成(薹草屬與禾本科的比例)顯著相關(guān)�？梢�,遺傳和食物都會(huì)影響雁腸道微生物的結(jié)構(gòu)。雁腸道微生物基本組成及影響因素研究,為揭示腸道微生物在水鳥遷徙與適應(yīng)過程中的作用提供了重要參考。4.草食性雁腸道微生物互作與功能微生物之間的相互作用不僅是微生物多樣性的重要組成部分,同時(shí)也會(huì)影響其群落功能的執(zhí)行�；�16S數(shù)據(jù),本研究引入RMT(random matrix theory,隨機(jī)矩陣?yán)碚?模型,構(gòu)建雁腸道微生物分子生態(tài)網(wǎng)絡(luò)(molecular ecology networks)。結(jié)果表明,升金湖與鄱陽湖的白額雁之間、鄱陽湖三種雁之間,在分子生態(tài)網(wǎng)絡(luò)的結(jié)構(gòu)、節(jié)點(diǎn)特征等方面存在明顯差異：證實(shí)腸道微生物之間的互作具有較高的多樣性。同時(shí),我們引入PICRUSt(phylogenetic investigation of communities by reconstruction of unobserved states)對微生物功能進(jìn)行預(yù)測。發(fā)現(xiàn),所有樣品組的微生物都執(zhí)行一些保守的功能,主要是參與物質(zhì)和能量代謝的基因。這些研究主要基于16S測序數(shù)據(jù)進(jìn)行推測。微生物之間真正的相互作用以及功能發(fā)揮,仍需要功能基因組學(xué)等技術(shù)進(jìn)行直接解析。上述研究,1)首次構(gòu)建了安徽升金湖國家級(jí)自然保護(hù)區(qū)冬春季節(jié)濕地植物基因庫；2)引入了宏條形碼技術(shù)分析草食性雁的食性,并證實(shí)其可用性；3)首次綜合闡述多種雁腸道微生物組成與結(jié)構(gòu)；4)引入分子生態(tài)網(wǎng)絡(luò)和PICRUSt方法解析雁腸道微生物的互作與功能。本研究表明,交叉學(xué)科的應(yīng)用可以實(shí)現(xiàn)對雁等鳥類的深入認(rèn)識(shí)和理解。本論文的主要?jiǎng)?chuàng)新點(diǎn)為：1)在方法學(xué)上,對傳統(tǒng)的食性分析方法進(jìn)行改善；2)在研究內(nèi)容上,闡述了多種雁的雁腸道微生物結(jié)構(gòu)與功能,并解析了其與食物的相關(guān)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.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q958

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本文編號(hào)：1663247