细菌与甲烷菌在牦牛瘤胃中的时间动态及其在消化道的空间分布

发布时间：2018-06-16 09:28

  本文选题：牦牛 + 细菌　； 参考：《兰州大学》2016年博士论文

【摘要】：青藏高原被称为世界“第三极”,拥有全球海拔最高、面积最大和唯一被四季放牧利用的高寒草地。独特的高寒生态环境(高海拔、寒冷、缺氧、紫外线强、牧草季节供应严重失衡),使得生存于此的牦牛种群在协同进化过程中产生了一系列特殊的适应机制。近年来的研究发现,与黄牛相比,牦牛在消化器官组织结构、牧食行为、氮素利用效率、甲烷排放、瘤胃甲烷菌菌群结构、季节间能量分配等方面均优于黄牛;并从牦牛全基因组测序中找到了适应高寒营养胁迫的相关功能基因。为此我们推断,长期的极端环境与营养胁迫,使细菌/甲烷菌在牦牛瘤胃和不同肠道部位中分别形成了特殊的时间动态和空间分布模式,以帮助宿主提高能量利用效率和有效应对每年长达8个月的冷季营养匮乏。本试验以牦牛为研究对象,主要利用通用引物515F/806R通过16S二代高通量测序,分析了细菌/甲烷菌在瘤胃中的定植过程(3、14、60、180、365和730天)、季节动态(春、夏、冬)和不同肠道部位(19个部位)的空间分布,拟寻找牦牛肠道微生态系统中有助于宿主适应高寒严酷生态系统的特殊微生物群落,为进一步通过调控肠道微生态系统提高牦牛生产性能和实现藏区畜牧业的可持续发展提供理论基础和技术支撑。本研究得到的主要结果如下:1.牦牛瘤胃细菌/甲烷菌定植过程中丰度指数Chao1的增加分为三个梯度,3和14天为第一阶段,60、180和365天为第二阶段,730天为第三阶段;多样性指数Shannon在3、14天低于其它年龄组(P0.05),其它年龄组之间没有差异(P0.05)。2.牦牛瘤胃细菌定植过程中优势门水平的细菌是厚壁菌门(Firmicutes),拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)三大类,约占85%。其中厚壁菌门(Firmicutes)所占的比例最高,在不同年龄组之间维持在约50%;拟杆菌门(Bacteroidetes)从3天的23%增加到了730天的43%;变形菌门(Proteobacteria)从3天的17%降低到了730天的2.3%。以上两种菌门的增加/降低都呈现出幼龄段(3、14天)、发育段(60、180天)和成年段(365、730天)阶梯式的变化。3.牦牛瘤胃甲烷菌—广古菌门从3天的0.3%增加到60天最高的2%,随后在成年组(365、730天)维持在约1.3%。4.瘤胃微生物定植过程中的途径主要是通过母畜唾液,将从母畜瘤胃中反刍带到口腔的瘤胃微生物转移到幼龄反刍动物的瘤胃中。5.牦牛瘤胃细菌/甲烷菌季节变化过程中的丰度指数Chao1从大到小的顺序是春夏秋,多样性指数Shannon与丰度指数呈现相同的趋势,但是春夏两季之间多样性更加接近。同一季节组内相似性春季最低,夏季最高。6.不同季节间以厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌,在春、夏、冬三个季节所占的比例分别为75%、80%和77%。其中拟杆菌门的组成比例在春季最低约45%,在夏冬两季基本相同约为54%;而厚壁菌门在春夏冬三个季节的比例分别是30、26、23%。7.在冷季(春冬两季),由于牦牛采食牧草中的纤维含量增高,瘤胃微生物中分解纤维的菌属高于暖季(夏季),而夏季中降解植物次级代谢产物和利用可溶性糖、氨基酸的菌属高于冷季。春季中的纤维降解菌属的种类和比例高于冬季。牦牛瘤胃甲烷菌的比例在冬季最低,而春夏两季的比例基本相同。8.在相同的低氮日粮下,牦牛和黄牛不同肠道部位的细菌/古菌Alpha和Beta多样性没有差异。细菌/甲烷菌在不同肠道部位的丰度指数(Chao1)和多样性指数(Shannon),都是在前肠道内容物和大肠最高,前肠道壁次之,小肠中最低。不同肠道部位同一样品组内微生物多样性在小肠部位最低,其次是大肠,前肠道(内容物和肠道壁)中相似性最高。9.忽略不同的肠道部位,在门水平的优势细菌是厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),在前肠道和小肠约占75%,而大肠中的占比高达90%。厚壁菌门(Firmicutes)在小肠和大肠中的比例高于前肠道,而拟杆菌门(Bacteroidetes)在前肠道部位高于小肠和大肠。甲烷菌的比例在小肠高于肠道其它部位。Cyanobacteria菌门在牦牛不同肠道部位的比例高于黄牛。10.在前肠道壁分布的优势菌属主要参与氧气、尿素和挥发性脂肪酸的吸收利用;前肠道部位内容物中比例较高的菌属主要负责来自日粮中的纤维素、植物次级代谢产物、淀粉和糖类的降解与利用;小肠中具有优势的菌属很少,主要是参与维生素的合成和黏素的降解;大肠主要负责吸收和利用一些没有被前肠道部位消化吸收的糖类、次级代谢产物和盐类。PICRUSt功能分析显示牦牛肠道微生态中比黄牛存在更多未知的菌群。与KEGG数据库比对,牦牛高于黄牛的基因家族的数量要多于黄牛高于牦牛的数量,其中能量储存、脂质代谢和聚糖合成和代谢的三大基因家族在牦牛要高于黄牛,这些基因家族的差异,可能会帮助牦牛提高能量利用效率。本试验首次全面分析了细菌/甲烷菌在放牧牦牛瘤胃中的时间动态和不同肠道部位的空间分布。同时,利用PICRUSt对细菌/甲烷菌的基因功能进行了全面预测,为青藏高原极端环境条件下的牦牛肠道微生物如何应对严酷的环境胁迫提供了重要的科学依据和理论支撑。
[Abstract]:The Qinghai Tibet Plateau is known as the "third pole" of the world. It has the highest altitudes, the largest area and the only cold grassland used by the four seasons. The unique Alpine ecological environment (high altitude, cold, anoxia, ultraviolet light, and the seasonal supply of herbage is out of balance), which makes a series of yak populations living in the process of co evolution. In recent years, studies have found that yaks are superior to yellow cattle in the structure of the digestive organs, grazing behavior, nitrogen use efficiency, methane emission, rumen methanogens group structure, and interseasonal energy distribution, compared with yellow cattle, and the related functions adapted to alpine nutrition stress are found in the whole gene group of yak. For this reason, we infer that long-term extreme environment and nutritional stress make bacteria / methanogens in the rumen and different intestinal parts of the yak, respectively, to form a special time dynamic and spatial distribution pattern to help the host improve the efficiency of energy utilization and to respond effectively to the cold season nutrition shortage for up to 8 months. The main purpose of this study is to analyze the spatial distribution of bacterial / methanogens in the rumen of the rumen (3,14,60180365 and 730 days), seasonal dynamics (spring, summer, winter) and different intestinal sites (19 parts) by high throughput sequencing of the 16S two generation by the universal primer 515F/806R, which is intended to help the host adapt to the adaptation of the host to Gao Hanyan. The special microbial community of the cool ecosystem provides the theoretical basis and technical support for further regulating the production performance of the yaks and realizing the sustainable development of the Tibetan animal husbandry by regulating the intestinal microflora system. The main results of this study are as follows: 1. the increase of the abundance index Chao1 in the rumen bacteria / methane bacteria colonization process of the yak is divided into two parts. The three gradient, 3 and 14 days were the first, the 60180 and 365 days were second, the 730 day was third; the diversity index Shannon was lower than the other age groups (P0.05), and there was no difference between the other age groups (P0.05) the bacteria of the dominant gate level in the rumen bacterial colonization of.2. yak were Firmicutes, Bacter Oidetes) and deformable bacteria gate (Proteobacteria) were the three major categories, accounting for about 85%., which accounted for the highest proportion of the Firmicutes, maintained about 50% in different age groups, and the bacteriobacteria (Bacteroidetes) increased from 23% in 3 days to 43% in 730 days; the Proteus gate (Proteobacteria) decreased from 17% to two bacteria of 730 days from 3 days. The increase / decrease of the door showed a young age (3,14 days), the development segment (60180 days) and the adult segment (365730 days) of the step change of the.3. yak rumen methanogens - the palaeo - palaebacterium gate increased from 0.3% to the highest 60 days in the 3 day, and the pathway in the adult group (365730 days) to maintain in the about 1.3%.4. rumen microbial colonization was mainly through the mother. The abundance index of the.5. yak in the rumen of the rumen of the ruminant from the rumen of the animal to the rumen of the rumen to the rumen of the young ruminant is the same in the order of spring and summer autumn, the diversity index Shannon and the abundance index are the same, but between the two seasons of spring and summer. In the same season, the similarity in the same season is the lowest in spring, and the highest.6. in summer is Firmicutes and Bacteroidetes. In spring, summer and winter, the proportion of the three seasons is 75%, 80% and 77%., and the proportion of the phyllillas is about 45% in spring, and in the two seasons of summer and winter. The same agreement was 54%, while the proportion of the three seasons in the spring and summer winter was 30,26,23%.7. in the cold season (in the spring and winter two seasons). The fiber content in the rumen microbes was higher than that in the warm season (in summer), and the secondary metabolites of plants and the use of soluble sugar and amino acids in summer were higher than those in the warm season (in summer). The species and proportion of fiber degrading bacteria in spring are higher than that in winter. The proportion of yak rumen methanogens is the lowest in winter, and the proportion of the two seasons in spring and summer is basically the same as.8. in the same low nitrogen diet. There is no difference in the diversity of bacteria / Alpha and Beta in the different intestinal parts of yak and yellow cattle. Bacteria / methanogens are different The abundance index (Chao1) and diversity index (Shannon) of the intestinal tract were the highest in the anterior intestine and the large intestine. The anterior intestinal wall was the second and the lowest in the small intestine. The microbial diversity in the same sample group was the lowest in the small intestine, followed by the large intestine, and the highest similarity in the anterior intestine (content and intestinal wall) was ignored by.9.. In the intestinal tract, the dominant bacteria at the portal level are Firmicutes and Bacteroidetes, in the anterior intestine and the small intestine about 75%, while the proportion of the large intestine in the large intestine is higher than that in the small intestine and large intestine (Firmicutes) in the small intestine and the large intestine than in the anterior intestine, and the bacteribacillus gate (Bacteroidetes) is higher in the anterior intestine than in the small intestine and the large intestine (Bacteroidetes). The proportion of methanogens in the small intestine is higher than that of.Cyanobacteria bacteria in the other parts of the intestines. The proportion of.10. in the yak is higher than that of the yellow cattle. The dominant bacteria in the anterior intestinal tract are mainly involved in the absorption and utilization of oxygen, urea and volatile fatty acids; the bacteria with higher proportion in the contents of the anterior intestine are mainly responsible for the diet. The degradation and utilization of cellulose, secondary metabolites of plants, starch and carbohydrates; the dominant bacteria in the small intestine are rare, mainly involved in the synthesis of vitamins and the degradation of the amyloid; the large intestine is mainly responsible for absorption and utilization of some sugars that are not digested and absorbed by the anterior intestine. Secondary metabolites and salt.PICRUSt functions are analyzed. Compared with the KEGG database, the number of yaks higher than the yellow cattle is more than the number of yak, and the three big gene families of energy storage, lipid metabolism and glycan synthesis and metabolism in Yak cattle are higher than the yellow cattle, and the difference of these gene families can be found. It can help yak to improve the efficiency of energy utilization. The time dynamics and spatial distribution of bacteria / methanogens in the rumen of Grazing Yaks were analyzed for the first time. At the same time, the genetic function of bacteria / methanogens was comprehensively predicted by PICRUSt for the Yak's intestinal microbiology under the extreme environment of the Qinghai Tibet Plateau. How to deal with severe environmental coercion provides important scientific basis and theoretical support.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823.85
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本文编号：2026201