围封年限对内蒙古草地甲烷氧化菌群落结构和活性的影响

发布时间：2018-04-06 23:12

本文选题：克隆文库　切入点：草地　出处：《西北农林科技大学》2015年硕士论文

【摘要】：大气中的温室气体主要有二氧化碳(CO2)、甲烷(CH4)、和氧化亚氮(N2O)。虽然CO2在大气中的含量很多,但是单分子CH4的增温潜势是CO2的20-30倍,对全球变暖的贡献率很高。甲烷的源很多,汇主要有两个,一部分是在大气中通过光化学反应被羟基氧化,另一部分是在通气良好的土壤中被甲烷氧化菌氧化。草地土壤疏松,是甲烷的一个重要的汇,甲烷氧化菌的含量和种类都很丰富。内蒙古草地作为我国典型的温带半干旱草地,目前有很多研究集中在该草地甲烷通量及放牧对甲烷吸收的影响,只有少数的研究集中在放牧对甲烷氧化菌群落结构及其活性的影响。因此本论文主要研究不同围封年限甲烷氧化菌的群落结构及甲烷氧化菌的丰度,并试图找出甲烷氧化菌群落结构与甲烷通量之间的关系。在本研究中选择的样地为中国锡林河流域附近的内蒙古草地。三个样地分别是79E(草地从1979年开始围封),99E(草地从1999年围封),G(自由放牧草地)。三个样地土壤基因组DNA提取后,一方面采用巢氏扩增的方法对甲烷氧化菌的功能基因pmo A用特异性引物进行扩增,使用克隆文库的方法对PCR产物进行定性分析;另一方面,使用定量PCR的方法对三个样地中甲烷氧化菌的丰度进行定量分析。同时,分别在18°C和28°C分别对三个土样进行培养,计算甲烷氧化菌的活性。结果显示:在三个样地中,甲烷氧化菌的主要类群是Type I,Type II类型的甲烷氧化菌只有一个基因型且只出现在G样地中。OTU1存在于三个样地中且只有OTU1属于USC-γ。聚类分析表明,随着围封时间的增加,99E的甲烷氧化菌群落结构首先与79E聚在一起,然后和G聚在一起。和之前的研究结果比较说明随着围封年限的增加,甲烷氧化菌的群落结构发生了转变。甲烷氧化菌的丰度用每克干土pmo A基因的拷贝数来代表,定量分析结果表明三个样地的甲烷氧化菌丰度呈现这样一种趋势G(4.5×107)99E(2.8×107)79E(2.0×107)。说明随着围封年限的增加甲烷氧化菌的丰度降低,同时发现在三个样地中USC-γ类群的甲烷氧化菌的丰度最大。从培养实验可以看出,当温度升高时,甲烷氧化菌的活性增大,三个样地中,79E甲烷氧化菌活性对温度的敏感性最低,说明79E的甲烷氧化菌更能适应未来多变的环境。99E甲烷氧化菌活性对温度的敏感性最高,可能是由于99E处于过渡阶段,微生物之间的竞争可能导致甲烷氧化菌的活性比较敏感,但是随着围封年限的增加,99E中甲烷氧化菌的活性会逐渐趋于稳定,和79E的甲烷氧化菌活性相似。G样地中甲烷氧化菌的活性也较高,虽然在某些条件下甲烷氧化菌的活性很高,但是还存在很大的不确定性。综上所述,在未来全球变暖的情况下,79E甲烷氧化菌活性更稳定,更能适应环境的波动。对于三个样地,未来全球变暖三个样地汇的功能都会增强。
[Abstract]:The main greenhouse gases in the atmosphere are CO _ 2, Ch _ 4, and N _ 2O _ 2.Although the content of CO2 in the atmosphere is much, the temperature increasing potential of monolayer CH4 is 20-30 times of that of CO2, and the contribution to global warming is very high.There are two main sources of methane, one is oxidation of hydroxyl group through photochemical reaction in the atmosphere, the other is oxidation of methane oxidizing bacteria in well-ventilated soil.Loose grassland soil is an important sink of methane, methane oxidizing bacteria content and species are very rich.Inner Mongolia grassland is a typical semi-arid grassland in temperate zone in China. At present, many studies have focused on the effects of methane flux and grazing on methane absorption in Inner Mongolia grassland.Only a few studies focused on the effects of grazing on the community structure and activity of methanogen.Therefore, this paper mainly studies the community structure and abundance of methane oxidizing bacteria in different envelop years, and tries to find out the relationship between methane oxidizing bacteria community structure and methane flux.The sample plots selected in this study are Inner Mongolia grassland near the Xilin River Basin in China.The three plots are 79e (grassland has been enclosed since 1979) (grassland has been enclosed from 1999 to 1999).After extracting genomic DNA from soil of three sample plots, on the one hand, pmo A, a functional gene of methanoxidism, was amplified by specific primers, and PCR products were qualitatively analyzed by clone library, on the other hand,The abundance of methane-oxidizing bacteria in three plots was quantitatively analyzed by quantitative PCR.At the same time, three soil samples were cultured at 18 掳C and 28 掳C, respectively.The results showed that the main taxa of methanophiles in the three plots were Type ItType II, which had only one genotype. OTU1 existed only in G sample and only OTU1 belonged to USC- 纬.The results of cluster analysis showed that with the increase of encapsulation time, the community structure of methanoxidism was first clustered with 79E and then with G.Compared with the previous studies, the community structure of methanogens changed with the increase of envelop life.The abundance of methane-oxidizing bacteria was represented by the copy number of pmo A gene per gram dry soil. The quantitative analysis showed that the abundance of methanogen oxidized bacteria in the three plots showed a trend of 4.5 脳 107)99E(2.8 脳 107)79E(2.0 脳 107.The results showed that the abundance of methanoxy bacteria decreased with the increase of envelop life, and it was found that the abundance of USC- 纬 group was the largest in the three plots.It can be seen from the culture experiment that when the temperature increases, the activity of methanophore increases, and the activity of methanophore is the least sensitive to temperature in the three plots.It is suggested that the methanophore of 79E is more sensitive to temperature than that of the methanophile bacteria in the future, which may be due to the fact that 99E is in the transition stage, and the competition among the microorganisms may lead to the more sensitive activity of the methanoxy bacteria.However, with the increase of envelop life, the activity of methanogen bacteria will become stable gradually, which is similar to that of 79E. G sample is also higher, although the activity of methanogen bacteria is very high under some conditions.But there is still a lot of uncertainty.In conclusion, in the future global warming, the activity of methanophore is more stable and can adapt to the fluctuation of environment.For the three plots, the future global warming will enhance the function of the three sample sinks.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S812.2

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