本文選題：黃河入�？� + nirK�。� 參考：《山東農(nóng)業(yè)大學》2017年碩士論文

【摘要】：河口位于海陸交匯的地帶,其各種生物地球化學過程之間的相互作用十分活躍。氮是組成生物有機體的主要元素,也是影響河口富營養(yǎng)化發(fā)生和演變的關鍵生源要素之一。黃河入�？谖挥邳S河、渤海灣和萊州灣的交匯處,是勝利油田的所在地,黃河攜帶的大量泥沙、陸源污染物以及周邊人類活動的影響使得該地區(qū)生態(tài)環(huán)境極為復雜多變。目前大量人為氮素的輸入使黃河入海口環(huán)境日益惡化,使其承受著巨大的生態(tài)壓力。反硝化被認為是去除活性氮的關鍵過程。本研究以黃河入�？�5個典型采樣點(SA-SE)的沉積物樣品為研究對象,利用克隆文庫和定量PCR的方法研究兩種基因型反硝化細菌的多樣性、群落結(jié)構(gòu)及豐度,結(jié)合理化因子進行相關性分析,揭示環(huán)境因子與反硝化細菌群落的關系。主要研究結(jié)果如下:(1)以nirK、nirS基因為分子標記,建立克隆文庫,得到陽性克隆子并測序,獲得nirK基因有效序列370條,nirS基因有效序列356條。將序列按照95%的相似性進行OTU(操作分類單元)劃分,nirK基因共獲得120個OTUs,nirS基因共獲得126個OTUs。對于nirK基因,每個采樣點的OTUs個數(shù)在19-55之間,nirS基因各個采樣點OTUs的個數(shù)在30-39之間。(2)基于OTU水平的α-多樣性分析表明,入海口nirS型反硝化細菌多樣性整體略高于nirK型反硝化細菌多樣性,且無論nirK或nirS基因,均表現(xiàn)為采樣點SE反硝化細菌多樣性較高。基于OTU水平的β-多樣性分析結(jié)果表明,兩種基因型的反硝化細菌其物種組成的空間分布方式不同,對nirK型反硝化細菌,SB和SE點的物種組成相似,而SA和SE點的nirS型反硝化細菌物種組成較相似。(3)NirK基因系統(tǒng)發(fā)育分析表明,與絕大部分OTUs親緣關系較近的克隆序列主要來自黃河入海口水體、舊金山灣河口沉積物、重金屬污染的土壤、活性污泥、沿海微生物墊和各種農(nóng)田土等環(huán)境。NirK基因系統(tǒng)進化樹可劃分為7個Clusters(Cluster I-VII),其中,Cluster I、III、IV、V的絕大部分序列主要分布在溶解氧含量較低的采樣點(SB、SC、SE),其他Clusters則分布在溶解氧含量較高的采樣點(SA、SD)。NirS基因系統(tǒng)發(fā)育分析表明,與絕大部分OTUs親緣關系較近的克隆子主要來自長江入海口、東湖、海河、北運河、珠江口和切薩皮克灣等河口沉積物環(huán)境,nirS基因系統(tǒng)進化樹可劃分為5個Clusters(Cluster I-V),與nirK型反硝化細菌群落結(jié)構(gòu)不同,nirS型反硝化細菌群落結(jié)構(gòu)不存在類似的空間分布模式。(4)利用定量PCR估算nirK和nirS型反硝化細菌豐度,nirK型反硝化細菌豐度為4.17×103-9.72×105 copies/g(干重),nirS型反硝化細菌豐度為4.34×104-2.62×105copies/g(干重)。單因素方差分析顯示,樣品SA、SC和SD中的nirS型反硝化細菌豐度顯著地高于其他采樣點(P0.05)。對于nirK,樣本間的基因豐度顯著不同(P0.001)。對兩種基因型反硝化細菌豐度比較分析,相比nirS,nirK型反硝化細菌在潮汐區(qū)域(SA、SC和SD)顯示較高的豐度,在河流匯合點(SB和SE)豐度較低。(5)環(huán)境因子與兩種基因型反硝化細菌的多樣性、豐度及優(yōu)勢OTUs的斯皮爾曼(Spearman)相關性分析表明,相比nirK型反硝化細菌,黃河入�？诔练e物nirS型反硝化細菌的多樣性及豐度對環(huán)境因子的響應更為敏感�；趦�(yōu)勢OTUs與全部OTUs的典型對應分析(Canonical Correspondence Analysis,CCA)表明,nirK型反硝化細菌群落結(jié)構(gòu)對環(huán)境因子的響應較為敏感,即nirK型反硝化細菌群落結(jié)構(gòu)受到溶解氧的顯著影響(P0.05),而nirS型反硝化細菌群落結(jié)構(gòu)受環(huán)境因子影響較小,可能主要受其他未被檢測到的環(huán)境因子所影響。
[Abstract]:The estuary is located in the area where the sea and land converge. The interaction between various biogeochemical processes is very active. Nitrogen is one of the main elements to form a biological organism. It is also one of the key factors that affect the occurrence and evolution of eutrophication in the estuary. The Yellow River entrance to the sea is located in the Yellow River, Bohai Bay and Laizhou Bay, which is a Shengli Oilfield. The impact of a large amount of sediment, land source and surrounding human activities in the Yellow River makes the ecological environment in the region very complex and changeable. A large amount of artificial nitrogen input makes the environment of the Yellow River entrance to the sea increasingly worsening, making it bear great ecological pressure. Denitrification is considered as a key process to remove active nitrogen. Based on the sediment samples of 5 typical sample points (SA-SE) in the Yellow River entrance to the sea, the diversity, community structure and abundance of two genotypic denitrifying bacteria were studied by cloning library and quantitative PCR, and the relationship between the environmental factors and denitrifying bacteria community was revealed. The results are as follows: (1) take nirK and nirS gene as molecular marker, establish clone library, obtain positive clones and sequence, obtain 370 effective sequence of nirK gene and 356 effective sequence of nirS gene. The sequence according to the similarity of 95% is divided into OTU (operation classification unit), nirK gene obtains 120 OTUs, nirS gene obtains 126 OTUs. for nirK. The number of OTUs at each sampling point is 19-55 and the number of OTUs at each sampling point of the nirS gene is 30-39. (2) the OTU level based alpha diversity analysis shows that the diversity of nirS denitrifying bacteria at the entrance to the sea is slightly higher than that of the nirK denitrifying bacteria, and the nirK or nirS genes are not discussed, all of which represent the SE denitrification finer at the sampling point. The beta diversity analysis based on OTU level showed that the spatial distribution of two genotypes of denitrifying bacteria was different, the species composition of nirK type denitrifying bacteria, SB and SE points was similar, while the species composition of nirS type denitrifying bacteria at SA and SE points was similar. (3) the phylogenetic analysis of NirK gene showed that the phylogenetic analysis of NirK gene showed that the species composition of denitrifying bacteria was similar. The most closely related clones of OTUs were mainly from the Yellow River entrance water, the sediments of San Francisco Bay Estuary, heavy metal contaminated soil, activated sludge, coastal microbial pads and various farmland soils could be divided into 7 Clusters (Cluster I-VII), including Cluster I, III, IV, V. Some sequences were mainly distributed in SB, SC, SE of low dissolved oxygen content, and other Clusters were distributed at the sampling point (SA, SD).NirS gene phylogenetic analysis of high dissolved oxygen content (SA, SD) showed that the clones closest to most OTUs were mainly from Changjiang River to Haikou, East Lake, Haihe River, North Canal, Pearl River mouth and Chesapeake The phylogenetic tree of the nirS gene could be divided into 5 Clusters (Cluster I-V), and the structure of the denitrifying bacteria community was different from that of nirK type denitrifying bacteria. (4) the abundance of nirK and nirS type denitrifying bacteria was estimated by quantitative PCR, and the abundance of nirK type denitrifying bacteria was 4.. 17 x 103-9.72 x 105 copies/g (dry weight), nirS type denitrifying bacteria abundance was 4.34 x 104-2.62 x 105copies/g (dry weight). Single factor variance analysis showed that the abundance of nirS type denitrifying bacteria in SA, SC and SD was significantly higher than that of other sampling points (P0.05). For nirK, the abundance of genes between samples was significantly different (P0.001). Against two types of denitrification. Compared with nirS, nirK type denitrifying bacteria showed higher abundance in Tidal Regions (SA, SC and SD) and lower abundance in river junction (SB and SE). (5) the diversity, abundance and Spielman (Spearman) correlation analysis of environmental factors and two genotypic denitrifying bacteria showed that compared to nirK type denitrification. The diversity and abundance of nirS type denitrifying bacteria in the sediments of the sea entrance of the Yellow River were more sensitive to environmental factors. Based on the typical corresponding analysis of the dominant OTUs and all OTUs (Canonical Correspondence Analysis, CCA), the response of the nirK type denitrifying bacteria community structure to environmental factors was more sensitive, that is, nirK type denitrification. The bacterial community structure was significantly affected by dissolved oxygen (P0.05), while the nirS type denitrifying bacteria community structure was less affected by environmental factors, and may be affected mainly by other environmental factors that were not detected.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q938

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