免培养法对新疆热气泉土壤原核微生物多样性及生物地理的研究
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摘要
本文采用免培养方法研究了新疆热气泉土壤原核微生物多样性及生物地理。通过构建细菌和古菌16S rDNA克隆文库,分析新疆玛纳斯热气泉土壤细菌和古菌多样性。采用T-RFLP方法,分析新疆玛纳斯3眼和石河子1眼热气泉土壤硫氧化和硫循环菌多样性及其与环境因子、样点位置间关系。主要研究内容与实验结果如下:
1.从新疆玛纳斯热气泉土壤细菌16S rDNA克隆文库中,随机挑选170个阳性克隆,用Hae III限制性内切酶进行RFLP分析,经分型测序,构建系统发育树,得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes),其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5 kb),占序列总数的48%。结果表明热气泉土壤细菌多样性较低,但存在大量潜在细菌新种。
2.从新疆玛纳斯热气泉土壤古菌16S rDNA克隆文库中,随机挑选98个阳性隆子,用HhaⅠ限制性内切酶进行RFLP分析,经分型测序,构建系统发育树,共得到16个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析均归为泉古菌门(Crenarchaeota),隶属于该门的两个分类单元(GroupⅠ和GroupⅡ)。结果表明栖息于热气泉土壤古菌中泉古菌占绝对优势。
3.从新疆玛纳斯3眼热气泉和石河子市1眼热气泉土壤总DNA中扩增硫氧化和硫还原菌arp A基因后,进行T-RFLP分析,结合测定土壤环境因子参数,经聚类分析、主成分分析(PCA)和典型相关分析(CCA),结果表明土壤温度和pH值影响热气泉土壤硫氧化和硫还原菌多样性,即在一定范围内,土壤温度和pH值能预测土壤硫氧化和硫还原菌多样性,而与其它环境因子无关。
4.从新疆玛纳斯3眼热气泉和石河子市1眼热气泉土壤总DNA中扩增细菌16S rDNA后,进行T-RFLP分析,并结合测定土壤环境因子参数,经聚类分析、主成分分析(PCA)和典型相关分析(CCA),结果表明除温度和pH值外的土壤有机质含量、铵态氮和硝态氮含量等理化因子影响土壤细菌多样性,此外样点间地理位置差异也影响土壤细菌多样性。
In order to investigate the prokaryotic diversity and biogeography in the hot gas spring soils, Xinjiang by Culture-independent Approach, we constructed bacterial and archaeal 16S rDNA clone libraries and then analysed the bacterial and archaeal diversity of the hot gas spring in MaNasi country. The aprA gene products of sulfate-reducing and sulfur-oxidizing prokaryote and bacterial 16S rDNA genes were analyzed with Terminal-Restriction Fragment Length Polymorphisms method and then studied the relationship between the Shannon-index and environmental factors. The main results were as follows:
(1)170 positive clones were randomly sleceted from the Bacterial 16S rDNA clone library,then processed by restriction fragment length polymorphism(RFLP) method using HaeⅢ. Unique rDNA types clones were sequenced, analysed and then constructed phylogenetic tree. Twenty-nine operational taxonomic units (OTU) from 170 positive clones were found belonging to 6 phyla: Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria. Firmicutes (71%) was the absolutely dominant components of the soil bacterial community. 14 sequences (about 1.5 kb) from 29 OTUs showed less affiliation with known taxa(<97% sequence similarity).Soil bacterial diversity is low in the hot gas spring soil, but exit a large number of new unknown taxon in this environment.
(2) 98 positive clones were randomly sleceted from the Archaeal 16S rDNA clone library,then processed by restriction fragment length polymorphism(RFLP) method using HhaⅠ. Unique rDNA types clones were sequenced, analysed and then constructed phylogenetic tree.Sixteen operational taxonomic units (OTU) from 98 positive clones were found belonging to Crenarchaeota and divided two groups. Crenarchaeota was the absolutely dominant components of the soil bacterial community inhabited in the hot gas spring soils.
(3)The PCR products of aprA gene from four hot gas spring soils were analysed by Terminal-Restriction Fragment Length Polymorphisms (RFLP) method with two different kind of restricted enzyme(HaeⅢand HhaⅠ). Combined with the environmental parameters by statistical analysis, investigated the relationship between the biodiversity of sulfate-reducing and sulfur-oxidizing prokaryotes inhabitating in the hot gas spring soil and environmental factors, the location of the sampling sites. The results revealed that the biodiversity just only was related to the temperature and pH of soils. So we can forecast the the biodiversity sulfate-reducing and sulfur-oxidizing prokaryotes in soils according to the temperature and pH of the four hot gas springs soils in a certain range.
(4)The products of bacterial 16S rDNA from four hot gas spring soils were analysed by Terminal-Restriction Fragment Length Polymorphisms (RFLP) method with two different kind of restricted enzyme(RsaⅠand HhaⅠ).Combined with the environmental parameters by statistical analysis, investigated the relationship between the biodiversity inhabitating in the hot gas spring soil and environmental factors, the location of the sampling sites. The results revealed that the biodiversity of bacteria was related to the environmental factors except temperature and pH.
1.从新疆玛纳斯热气泉土壤细菌16S rDNA克隆文库中,随机挑选170个阳性克隆,用Hae III限制性内切酶进行RFLP分析,经分型测序,构建系统发育树,得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes),其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5 kb),占序列总数的48%。结果表明热气泉土壤细菌多样性较低,但存在大量潜在细菌新种。
2.从新疆玛纳斯热气泉土壤古菌16S rDNA克隆文库中,随机挑选98个阳性隆子,用HhaⅠ限制性内切酶进行RFLP分析,经分型测序,构建系统发育树,共得到16个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析均归为泉古菌门(Crenarchaeota),隶属于该门的两个分类单元(GroupⅠ和GroupⅡ)。结果表明栖息于热气泉土壤古菌中泉古菌占绝对优势。
3.从新疆玛纳斯3眼热气泉和石河子市1眼热气泉土壤总DNA中扩增硫氧化和硫还原菌arp A基因后,进行T-RFLP分析,结合测定土壤环境因子参数,经聚类分析、主成分分析(PCA)和典型相关分析(CCA),结果表明土壤温度和pH值影响热气泉土壤硫氧化和硫还原菌多样性,即在一定范围内,土壤温度和pH值能预测土壤硫氧化和硫还原菌多样性,而与其它环境因子无关。
4.从新疆玛纳斯3眼热气泉和石河子市1眼热气泉土壤总DNA中扩增细菌16S rDNA后,进行T-RFLP分析,并结合测定土壤环境因子参数,经聚类分析、主成分分析(PCA)和典型相关分析(CCA),结果表明除温度和pH值外的土壤有机质含量、铵态氮和硝态氮含量等理化因子影响土壤细菌多样性,此外样点间地理位置差异也影响土壤细菌多样性。
In order to investigate the prokaryotic diversity and biogeography in the hot gas spring soils, Xinjiang by Culture-independent Approach, we constructed bacterial and archaeal 16S rDNA clone libraries and then analysed the bacterial and archaeal diversity of the hot gas spring in MaNasi country. The aprA gene products of sulfate-reducing and sulfur-oxidizing prokaryote and bacterial 16S rDNA genes were analyzed with Terminal-Restriction Fragment Length Polymorphisms method and then studied the relationship between the Shannon-index and environmental factors. The main results were as follows:
(1)170 positive clones were randomly sleceted from the Bacterial 16S rDNA clone library,then processed by restriction fragment length polymorphism(RFLP) method using HaeⅢ. Unique rDNA types clones were sequenced, analysed and then constructed phylogenetic tree. Twenty-nine operational taxonomic units (OTU) from 170 positive clones were found belonging to 6 phyla: Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria. Firmicutes (71%) was the absolutely dominant components of the soil bacterial community. 14 sequences (about 1.5 kb) from 29 OTUs showed less affiliation with known taxa(<97% sequence similarity).Soil bacterial diversity is low in the hot gas spring soil, but exit a large number of new unknown taxon in this environment.
(2) 98 positive clones were randomly sleceted from the Archaeal 16S rDNA clone library,then processed by restriction fragment length polymorphism(RFLP) method using HhaⅠ. Unique rDNA types clones were sequenced, analysed and then constructed phylogenetic tree.Sixteen operational taxonomic units (OTU) from 98 positive clones were found belonging to Crenarchaeota and divided two groups. Crenarchaeota was the absolutely dominant components of the soil bacterial community inhabited in the hot gas spring soils.
(3)The PCR products of aprA gene from four hot gas spring soils were analysed by Terminal-Restriction Fragment Length Polymorphisms (RFLP) method with two different kind of restricted enzyme(HaeⅢand HhaⅠ). Combined with the environmental parameters by statistical analysis, investigated the relationship between the biodiversity of sulfate-reducing and sulfur-oxidizing prokaryotes inhabitating in the hot gas spring soil and environmental factors, the location of the sampling sites. The results revealed that the biodiversity just only was related to the temperature and pH of soils. So we can forecast the the biodiversity sulfate-reducing and sulfur-oxidizing prokaryotes in soils according to the temperature and pH of the four hot gas springs soils in a certain range.
(4)The products of bacterial 16S rDNA from four hot gas spring soils were analysed by Terminal-Restriction Fragment Length Polymorphisms (RFLP) method with two different kind of restricted enzyme(RsaⅠand HhaⅠ).Combined with the environmental parameters by statistical analysis, investigated the relationship between the biodiversity inhabitating in the hot gas spring soil and environmental factors, the location of the sampling sites. The results revealed that the biodiversity of bacteria was related to the environmental factors except temperature and pH.
引文
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[2]吴继尧,姚华,郑玉建.新疆热气泉调查分析[J].新疆医学院学报(Acta Academiae Medicinae Xin Jiang). 1994,17(2):93-96.
[3]新疆地方志编纂委员会.新疆通志·地质矿产志[M].新疆乌鲁木齐:新疆人民出版社,1997.
[4] Dion P,Nautiyal CS. Microbiology of Extreme Soils. Germany,2008.
[5] Norris TB, Wraith JM, Castenholz RW, et al. Soil microbial community structure across a thermal gradient following a geothermal heating event[J]. Applied And Environmental Microbiology,2002,68:6300–6309.
[6] Yeager CM, Northup DE, Grow CC, et al. Changes in nitrogen-fixing and ammonia-oxidizing bacterial communities in soil[J]. Applied And Environmental Microbiology,2005,71:2713–2722.
[7] Tobin-Janzen T,Shade A,Marshall L,et al. Nitrogen changes and domain bacteria ribotype diversity in soils overlying the Chentralia,Pennsylvania underground coal mine fire[J]. Soil science,2005,170(3):191-201.
[8] Ramette A,Tiedje JM. Biogeography:An Emerging Cornerstone for Understanding Prokaryotic Diversity, Ecology, and Evolution[J]. Microbial Ecology,2007,53:197-207.
[9]宋福强,微生物生态学[M].北京:化学工业出版社,2008.
[10] Hughes-Martiny JB,Bohannan BJM,Brown JH,et al. Microbial biogeography:putting microorganisms on the map[J]. Nature,2006,4:102-112.
[11] Fulthorpe RR, Rhodes AN , Tiedje JM. High levels of endemicity of 3-chlorobenzoate-degrading soil bacteria[J]. Appl Environ Microbiol. 1998,64: 1620–1627.
[12] Cho JC,Tiedje JM.Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil[J]. Appl Environ Microbiol. 2000,66:5448–5456.
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