海洋浮霉状菌分子生态学研究
摘要
由于具有“细胞器”和缺少肽聚糖等特性,浮霉菌成为研究细菌和真核细胞之间进化关系的模式微生物,近年来已经是微生物中研究的热点之一。以前的工作显示浮霉菌在海洋碳和氮循环中起着重要的作用,如厌氧氨氧化过程对于海洋N循环中氮气的形成有30-70%的贡献率。虽然针对于浮霉菌的调查在不断的增加,但对于海洋环境中浮霉菌的分布、多样性及其丰度知道的还是非常有限。本论文主要涉及海洋浮霉菌的分子生态学研究,包括以下方面的工作:
1)本文应用16S rRNA基因的手段调查了南中国海-沉积物柱状样和西太平洋一纬度梯度的表层水样浮霉菌的组成。对于南中国海沉积物柱状样(7个层,包括0.1m,1m,3m,5m,7m,9m和11m),用Pla-46-F/1392-R进行的PCR扩增显示浮霉菌只存在于有限的层(≤5m),并且随着沉积物深度的增加浮霉菌的多样性下降,沉积物中所得到的浮霉菌的序列主要是Pirellula属的序列:对于西太平洋表层水,Pirellula-Rhodopirellula-Blastopirellula(PRB)是浮霉菌的最重要的组成,并具有较高的多样性,而其它属Gemmata和Planctomyces只发现于H5和H2站位,且低纬度的表层水中浮霉菌的多样性比中纬的要高些。应用∫-libshuff软件统计分析的结果显示沉积物和表层水浮霉菌的组成存在显著性差异,可能是受复杂的水文条件、各种不同的地化环境因子的影响。
2)设计并得到二条浮霉菌特异性后引物,在理论和实验上都证明所获得的新引物可以大大的提高浮霉菌特异性前引物的扩增能力,实验结果显示新引物可以在属的水平上补充浮霉菌的多样性;表明以前只用引物Pla-46F/1392-R对浮霉菌的调查存在低估的可能。
3)获得了南中国海沉积物0.1m层参与氨氧化的微生物的分子证据(包括厌氧氨氧化细菌的16S rRNA基因序列、β变形菌氨氧化细菌的16S rRNA基因序列及氨氧化古菌的氨单加氧基因amoA的序列),显示了沉积物中复杂的氨氧化微生物的组成,为认识海洋沉积物氨氧化微生物群落中复杂的竞争或协作的关系提供信息。
4)应用CARD-FISH技术揭示了长江口及三个南中国海剖面站位浮霉菌的丰度。虽然长江口浮霉菌的丰度(0.23-20.53×10~4个细胞/毫升)要高于南中国海水柱中的浮霉菌的丰度(0.15-10.25×10~3个细胞/毫升),但本研究的结果低于以前报道的在沉积物和土壤中浮霉菌的丰度。
In recent decades,Planctomycetes has become one of the focused groups,for Planctomycetes is a microbial model to explore the evolution relationship between bacteria and eukaryotes for its characteristic of compartment and peptidoglycan-less. Previous work showed that Planctomycetes played important roles in marine carbon and nitrogen cycles such as anammox processes attributed to 30-70%marine nitrogen gas losses.Although Planctomycetes investigations have been increased,little is known about the distribution,diversity and abundance of Planctomycetales regarding marine environments.Here we present some molecular works on marine Planctomycetes.
1) 16S rRNA gene approach is applied to investigate Planctomycetes community in the vertical deep sea sediment of the South China Sea and the latitludinal surface seawater of the Western Pacific Ocean.For the vertical sediment of the South China Sea,PCR amplification using the primer set Pla-46-F/1392-R of the samples from seven sediment layers(0.1m,1m,3m,5m,7m,9m and 11m below the surface sediment) showed that Planctomycetales existed within a limited range of sediment depths(≤5m), and had a decreasing tread in diversity with increasing depth.The majority of the Pla-46-F/1392-R retrieved sequences belong to Pirellula-related Planctomycetales,For the latitludinal surface seawater of the Western Pacific Ocean,the results revealed that Pirellula-Rhodopirellula-Blastopirellula clade dominated the Planctomycetes community(between 83.3%and 94.1%) in all surface seawater sites and was present unexpected diverse while the minority of genera Gemmata and Planctomyces were only found in sites H5 and H2 respectively.Interestingly,diversity of low latitudes seawater appeared higher than of mid-lantitudes,∫-libshuff software analysis revealed significantly different diversity pattern between in the latitudinal surface seawater and in the sediment,possibly response to different hydrological and geographic features
2) Two new reverse primers(Pla-1097-R and Pla-1368-R) are designed,which greatly promote the potential ability of the forward primer Pla-46-F,both theoretically and experimentally.The test showed that PCR using the new primer sets reduced mismatches to non-Planctomycetes and supplemented the diversity of Planctomycetes, especially increasing the numbers of genera of Planctomycetes in both seawater and sediment samples.Our results strongly suggest that the diversity of Planctomycetes as revealed by Pla-46-F/1392-R has been potentially underestimated.
3) Molecular evidences of ammonium oxidizing microorganisms were present in the 0.1m layer in the South China Sea sediment station MD2896,including anammox-related 16S rRNA gene sequences,beta-Proteobacteria ammonium oxidizing bacteria 16S rRNA gene sequences and archaea amoA gene sequences.Our data suggested that there are comprehensive competitions or cooperations among different ammonium oxidizing microorganisms in the sediment.
4) CARD-FISH approach was applied to investigate the abundance of Planctomycetes in China seas.Our data revealed that the abundance of Planctomycetes was varied 0.23-20.53×10~4 cells/ml in The Yangtze River estuary and 0.15-10.25×10~3 cells/ml in the seawater columns of the South China Sea respectively.Even though higher than in the seawater columns of South China Sea,the abundance of Planctomycetes in The Yangtze River estuary is lower than previous reported in soils and sediments.
1)本文应用16S rRNA基因的手段调查了南中国海-沉积物柱状样和西太平洋一纬度梯度的表层水样浮霉菌的组成。对于南中国海沉积物柱状样(7个层,包括0.1m,1m,3m,5m,7m,9m和11m),用Pla-46-F/1392-R进行的PCR扩增显示浮霉菌只存在于有限的层(≤5m),并且随着沉积物深度的增加浮霉菌的多样性下降,沉积物中所得到的浮霉菌的序列主要是Pirellula属的序列:对于西太平洋表层水,Pirellula-Rhodopirellula-Blastopirellula(PRB)是浮霉菌的最重要的组成,并具有较高的多样性,而其它属Gemmata和Planctomyces只发现于H5和H2站位,且低纬度的表层水中浮霉菌的多样性比中纬的要高些。应用∫-libshuff软件统计分析的结果显示沉积物和表层水浮霉菌的组成存在显著性差异,可能是受复杂的水文条件、各种不同的地化环境因子的影响。
2)设计并得到二条浮霉菌特异性后引物,在理论和实验上都证明所获得的新引物可以大大的提高浮霉菌特异性前引物的扩增能力,实验结果显示新引物可以在属的水平上补充浮霉菌的多样性;表明以前只用引物Pla-46F/1392-R对浮霉菌的调查存在低估的可能。
3)获得了南中国海沉积物0.1m层参与氨氧化的微生物的分子证据(包括厌氧氨氧化细菌的16S rRNA基因序列、β变形菌氨氧化细菌的16S rRNA基因序列及氨氧化古菌的氨单加氧基因amoA的序列),显示了沉积物中复杂的氨氧化微生物的组成,为认识海洋沉积物氨氧化微生物群落中复杂的竞争或协作的关系提供信息。
4)应用CARD-FISH技术揭示了长江口及三个南中国海剖面站位浮霉菌的丰度。虽然长江口浮霉菌的丰度(0.23-20.53×10~4个细胞/毫升)要高于南中国海水柱中的浮霉菌的丰度(0.15-10.25×10~3个细胞/毫升),但本研究的结果低于以前报道的在沉积物和土壤中浮霉菌的丰度。
In recent decades,Planctomycetes has become one of the focused groups,for Planctomycetes is a microbial model to explore the evolution relationship between bacteria and eukaryotes for its characteristic of compartment and peptidoglycan-less. Previous work showed that Planctomycetes played important roles in marine carbon and nitrogen cycles such as anammox processes attributed to 30-70%marine nitrogen gas losses.Although Planctomycetes investigations have been increased,little is known about the distribution,diversity and abundance of Planctomycetales regarding marine environments.Here we present some molecular works on marine Planctomycetes.
1) 16S rRNA gene approach is applied to investigate Planctomycetes community in the vertical deep sea sediment of the South China Sea and the latitludinal surface seawater of the Western Pacific Ocean.For the vertical sediment of the South China Sea,PCR amplification using the primer set Pla-46-F/1392-R of the samples from seven sediment layers(0.1m,1m,3m,5m,7m,9m and 11m below the surface sediment) showed that Planctomycetales existed within a limited range of sediment depths(≤5m), and had a decreasing tread in diversity with increasing depth.The majority of the Pla-46-F/1392-R retrieved sequences belong to Pirellula-related Planctomycetales,For the latitludinal surface seawater of the Western Pacific Ocean,the results revealed that Pirellula-Rhodopirellula-Blastopirellula clade dominated the Planctomycetes community(between 83.3%and 94.1%) in all surface seawater sites and was present unexpected diverse while the minority of genera Gemmata and Planctomyces were only found in sites H5 and H2 respectively.Interestingly,diversity of low latitudes seawater appeared higher than of mid-lantitudes,∫-libshuff software analysis revealed significantly different diversity pattern between in the latitudinal surface seawater and in the sediment,possibly response to different hydrological and geographic features
2) Two new reverse primers(Pla-1097-R and Pla-1368-R) are designed,which greatly promote the potential ability of the forward primer Pla-46-F,both theoretically and experimentally.The test showed that PCR using the new primer sets reduced mismatches to non-Planctomycetes and supplemented the diversity of Planctomycetes, especially increasing the numbers of genera of Planctomycetes in both seawater and sediment samples.Our results strongly suggest that the diversity of Planctomycetes as revealed by Pla-46-F/1392-R has been potentially underestimated.
3) Molecular evidences of ammonium oxidizing microorganisms were present in the 0.1m layer in the South China Sea sediment station MD2896,including anammox-related 16S rRNA gene sequences,beta-Proteobacteria ammonium oxidizing bacteria 16S rRNA gene sequences and archaea amoA gene sequences.Our data suggested that there are comprehensive competitions or cooperations among different ammonium oxidizing microorganisms in the sediment.
4) CARD-FISH approach was applied to investigate the abundance of Planctomycetes in China seas.Our data revealed that the abundance of Planctomycetes was varied 0.23-20.53×10~4 cells/ml in The Yangtze River estuary and 0.15-10.25×10~3 cells/ml in the seawater columns of the South China Sea respectively.Even though higher than in the seawater columns of South China Sea,the abundance of Planctomycetes in The Yangtze River estuary is lower than previous reported in soils and sediments.
引文
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