本文選題：克隆文庫(kù)　切入點(diǎn)：草地　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：大氣中的溫室氣體主要有二氧化碳(CO2)、甲烷(CH4)、和氧化亞氮(N2O)。雖然CO2在大氣中的含量很多,但是單分子CH4的增溫潛勢(shì)是CO2的20-30倍,對(duì)全球變暖的貢獻(xiàn)率很高。甲烷的源很多,匯主要有兩個(gè),一部分是在大氣中通過(guò)光化學(xué)反應(yīng)被羥基氧化,另一部分是在通氣良好的土壤中被甲烷氧化菌氧化。草地土壤疏松,是甲烷的一個(gè)重要的匯,甲烷氧化菌的含量和種類都很豐富。內(nèi)蒙古草地作為我國(guó)典型的溫帶半干旱草地,目前有很多研究集中在該草地甲烷通量及放牧對(duì)甲烷吸收的影響,只有少數(shù)的研究集中在放牧對(duì)甲烷氧化菌群落結(jié)構(gòu)及其活性的影響。因此本論文主要研究不同圍封年限甲烷氧化菌的群落結(jié)構(gòu)及甲烷氧化菌的豐度,并試圖找出甲烷氧化菌群落結(jié)構(gòu)與甲烷通量之間的關(guān)系。在本研究中選擇的樣地為中國(guó)錫林河流域附近的內(nèi)蒙古草地。三個(gè)樣地分別是79E(草地從1979年開(kāi)始圍封),99E(草地從1999年圍封),G(自由放牧草地)。三個(gè)樣地土壤基因組DNA提取后,一方面采用巢氏擴(kuò)增的方法對(duì)甲烷氧化菌的功能基因pmo A用特異性引物進(jìn)行擴(kuò)增,使用克隆文庫(kù)的方法對(duì)PCR產(chǎn)物進(jìn)行定性分析;另一方面,使用定量PCR的方法對(duì)三個(gè)樣地中甲烷氧化菌的豐度進(jìn)行定量分析。同時(shí),分別在18°C和28°C分別對(duì)三個(gè)土樣進(jìn)行培養(yǎng),計(jì)算甲烷氧化菌的活性。結(jié)果顯示:在三個(gè)樣地中,甲烷氧化菌的主要類群是Type I,Type II類型的甲烷氧化菌只有一個(gè)基因型且只出現(xiàn)在G樣地中。OTU1存在于三個(gè)樣地中且只有OTU1屬于USC-γ。聚類分析表明,隨著圍封時(shí)間的增加,99E的甲烷氧化菌群落結(jié)構(gòu)首先與79E聚在一起,然后和G聚在一起。和之前的研究結(jié)果比較說(shuō)明隨著圍封年限的增加,甲烷氧化菌的群落結(jié)構(gòu)發(fā)生了轉(zhuǎn)變。甲烷氧化菌的豐度用每克干土pmo A基因的拷貝數(shù)來(lái)代表,定量分析結(jié)果表明三個(gè)樣地的甲烷氧化菌豐度呈現(xiàn)這樣一種趨勢(shì)G(4.5×107)99E(2.8×107)79E(2.0×107)。說(shuō)明隨著圍封年限的增加甲烷氧化菌的豐度降低,同時(shí)發(fā)現(xiàn)在三個(gè)樣地中USC-γ類群的甲烷氧化菌的豐度最大。從培養(yǎng)實(shí)驗(yàn)可以看出,當(dāng)溫度升高時(shí),甲烷氧化菌的活性增大,三個(gè)樣地中,79E甲烷氧化菌活性對(duì)溫度的敏感性最低,說(shuō)明79E的甲烷氧化菌更能適應(yīng)未來(lái)多變的環(huán)境。99E甲烷氧化菌活性對(duì)溫度的敏感性最高,可能是由于99E處于過(guò)渡階段,微生物之間的競(jìng)爭(zhēng)可能導(dǎo)致甲烷氧化菌的活性比較敏感,但是隨著圍封年限的增加,99E中甲烷氧化菌的活性會(huì)逐漸趨于穩(wěn)定,和79E的甲烷氧化菌活性相似。G樣地中甲烷氧化菌的活性也較高,雖然在某些條件下甲烷氧化菌的活性很高,但是還存在很大的不確定性。綜上所述,在未來(lái)全球變暖的情況下,79E甲烷氧化菌活性更穩(wěn)定,更能適應(yīng)環(huán)境的波動(dòng)。對(duì)于三個(gè)樣地,未來(lái)全球變暖三個(gè)樣地匯的功能都會(huì)增強(qiáng)。
[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 擄C and 28 擄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- 緯.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- 緯 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S812.2

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本文編號(hào)：1719333