红树林根际土壤固氮细菌和功能基因nifH的多样性研究
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摘要
本研究利用阿须贝无氮培养基,从9种红树林植物根际土壤分离得到12种好氧自生固氮细菌,通过PCR扩增,获得全部菌株的16S rDNA序列以及9种菌株的nifH序列,根据这两种序列分别建立了系统发育树,比较显示,仅部分菌株在两种系统发育树中表现出相似的聚类结果,证明16S rRNA和nifH是两种进化速率不同的基因;并发现供试根际土壤可培养固氮细菌的优势类群分布于变形菌纲。进一步利用“PCR-克隆-测序”免培养法,从3种红树植物根际土壤克隆获得27种nifH序列,与有关文献公布的nifH序列一起建立了系统发育树。分析表明:在同一地域红树林生态系统的不同根际土壤样品中,固氮细菌的分布存在差异,在理化性质差异明显的土壤样品之间,这种差异更为显著;首次报道来自同一地域红树林生态系统中的nifH序列倾向于聚集在一起形成相对独立的簇。分析认为,红树林土壤固氮细菌的群落组成在大尺度间具有较强的独立性,其多样性和优势类群更依赖于具体的小尺度环境特性。
Twelve aerobic free living nitrogen-fixing bacteria were isolated from nine rhizosphere soil samples of mangrove trees. Twelve 16S rDNA and nine nifH sequences of isolates were obtained by PCR, respectively. Two kinds of phylogenetic trees were constructed basing on 16S rDNA and nifH sequences of the isolates and available in GenBank. Comparison of two phylognetic trees, it was found that the aligment is similar only for a few strains, providing evidence that the difference in rates of evolution between 16S rRNA and nifH gene. By using PCR-Clone-Sequencing, a culture-independent approach, twenty-seven nifH sequences were obtained from three rhizosphere soil samples of mangrove trees. Neighbor-joining phylogenetic tree of nifH was constructed basing on all nifH sequences from mangrove ecosystem in GenBank as well as nifH sequences obtained in this study. It demonstrated that nifH diversity varied among different soil samples within a mangrove ecosystem, especially among samples with distinct physico-chemical properties; while among different geographical mangrove ecosystem, this is the first report, that the nifH sequences obtained from the same area were inclined to cluster as an independent group.The results implied that the composition of diazotrophs community in mangrove soil is mainly dependent on the specific characteristics of enviromental sample, but poorly associated among geographically isolated mangrove ecosystem.
Twelve aerobic free living nitrogen-fixing bacteria were isolated from nine rhizosphere soil samples of mangrove trees. Twelve 16S rDNA and nine nifH sequences of isolates were obtained by PCR, respectively. Two kinds of phylogenetic trees were constructed basing on 16S rDNA and nifH sequences of the isolates and available in GenBank. Comparison of two phylognetic trees, it was found that the aligment is similar only for a few strains, providing evidence that the difference in rates of evolution between 16S rRNA and nifH gene. By using PCR-Clone-Sequencing, a culture-independent approach, twenty-seven nifH sequences were obtained from three rhizosphere soil samples of mangrove trees. Neighbor-joining phylogenetic tree of nifH was constructed basing on all nifH sequences from mangrove ecosystem in GenBank as well as nifH sequences obtained in this study. It demonstrated that nifH diversity varied among different soil samples within a mangrove ecosystem, especially among samples with distinct physico-chemical properties; while among different geographical mangrove ecosystem, this is the first report, that the nifH sequences obtained from the same area were inclined to cluster as an independent group.The results implied that the composition of diazotrophs community in mangrove soil is mainly dependent on the specific characteristics of enviromental sample, but poorly associated among geographically isolated mangrove ecosystem.
引文
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8.林鹏.中国红树林生态系统.北京:科学出版社,1997
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12.王胤,左平,黄仲琪,邹欣庆.海南东寨港红树林湿地面积变化及其驱动力分析.四川环境,2006,25(3):44-49
13.邢德峰,任南琪,宫曼丽.PCR-DGGE技术解析生物制氢反应器微生物多样性.环境科学,2005,26(3):
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16.袁三青,薛燕芬,高鹏,汪卫东,马延和.T-RFLP技术分析油藏微生物多样性.微生物学报,2007,47(2):290-294
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20.张银龙,林鹏.九龙江河口秋茄林及白骨壤红树林土壤特性研究.河南农业大学学报,1998,32(4):325-330
21.阎冰,洪葵,喻子牛.红树林土壤16S rDNA多样性的研究.[博士学位论文].武汉:华中农业大学,2006
22.杨建,洪葵.红树林土壤总DNA不同提取方法比较研究.生物技术通报,2006,366-371
23.庄铁诚,林鹏.九龙江口秋茄(Kandelia candel)红树林掉落叶自然分解与落叶腐解微生物的关系.植物生态学报,1992,16(1):17-25
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2.顾华杰,李玉祥,赵明文,潘根兴.几种水稻田土壤微生物总DNA提取方法的比较.江苏大学学报(医学版),2005,15(4):300-305
3.蓝福生,莫全辉,陈平等.广西海滩土壤资源及合理开发利用.自然资源报,1993,(4):26-32
4.龙寒,向伟,庄铁城,林鹏.红树林区微生物资源.生态学杂志,2005,24(6):696-702
5.李慧,张颖,苏振成,徐慧.沈抚石油污水灌区稻田土壤细菌遗传多样性-16S rDNA-PCR-DGGE分析.土壤学报,2006,43(6):
6.廖金凤.海南岛的红树林盐土/范航清,梁士楚.中国红树林研究与管理.北京:科学技术出版社,1995:1-50
7.李玫.底泥微生物在红树林生态系统中的作用.广州环境科学,2006:21(4):21-25
8.林鹏.中国红树林生态系统.北京:科学出版社,1997
9.林鹏,张瑜斌.九龙江口红树林土壤微生物的类群及抗菌活性.海洋学报,2005,27(3):133-141
10.李庆芳,章家恩,刘金苓,唐国玲.红树林生态系统服务功能研究综述.生态科学,2006,25(5):472-475
11.王岳坤,洪葵.红树林土壤因子对土壤微生物数量的影响.热带作物学报,2005,26(3):109-114
12.王胤,左平,黄仲琪,邹欣庆.海南东寨港红树林湿地面积变化及其驱动力分析.四川环境,2006,25(3):44-49
13.邢德峰,任南琪,宫曼丽.PCR-DGGE技术解析生物制氢反应器微生物多样性.环境科学,2005,26(3):
14.徐海,陈少波,张素霞,仇建标,黄晓林.红树林土壤基本特征及发展前景安徽农业科学,2008,36(4):1496-1497,1504
15.谢瑞红,周兆德.红树林生态系统及功能研究综述.2005,11(4):48-52
16.袁三青,薛燕芬,高鹏,汪卫东,马延和.T-RFLP技术分析油藏微生物多样性.微生物学报,2007,47(2):290-294
17.杨萍如,何金海,刘膝辉.红树林及其土壤.自然资源学报.1987.2(1):32-37
18.张瑜斌,庄铁诚,杨志伟,林鹏.海南东寨港红树林土壤微生物初探.生态学杂志,2001,20(1):63-64
19.张瑜斌,曹卉,庄铁诚,林鹏.红树林固氮微生物研究进展.海洋通报,2003,22(6):79-82
20.张银龙,林鹏.九龙江河口秋茄林及白骨壤红树林土壤特性研究.河南农业大学学报,1998,32(4):325-330
21.阎冰,洪葵,喻子牛.红树林土壤16S rDNA多样性的研究.[博士学位论文].武汉:华中农业大学,2006
22.杨建,洪葵.红树林土壤总DNA不同提取方法比较研究.生物技术通报,2006,366-371
23.庄铁诚,林鹏.九龙江口秋茄(Kandelia candel)红树林掉落叶自然分解与落叶腐解微生物的关系.植物生态学报,1992,16(1):17-25
24.张忠华,胡刚,梁士楚.广西红树林资源与保护.海洋环境科学,2007,26(3):275-279,282
25.Alexandre S R,Gabriela F D,Lucy S,Jan D V E.Genetic diversity of nifH gene sequences in Paenibacillus azotofixans strains and soil samples analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragment s.Appl Environ Microbiol,1998,64(8):2770-2779
26.Alongi D M.Bacterial productivity and microbial biomass in tropical mangrov sediments.Microbecol,1988,15:59-75
27.Anandak and Sridharkr.Diversity of endophytic fungi in the roots of mangrov es species on the West Coast of India.Can J Microbiol,2002,48(10):871-878
28.Bashan Y,Puente M E,Myrold D D.In vitro transfer of fixed nitrogen fro diazotrophic filamentous cyanobacteria to black mangrove seedlings.Mcrobiol Ecology,1998,26(3):165-170
29. Bürgmann H, Widmer F, Sigler W V, Zeyer J. New molecular screening tools for analysis of free living diazotrophs in soil. Appl Environ Microbiol, 2004, 70 (1): 240-247
30. Burgmann H, Meier S, Bunge M, Widmer F, Zeyer J. Effects of model root e xudates on structure and activity of a soil diazotrop community. Environm Mi crobi, 2005, 7(11): 1711-1724
31. Coelho M R, Vos M D, Carneiro N P, Marriel I E, Paiva E, Seldin L. Divers ity of nifH gene pool in the rhizosphere of two cultivars of sorghum (Sorghu m bicolor) treated with contrasting levels of nitrogen fertilizer. FEMS Microbi ol Lett, 2008, 279: 15-22
32. Deslippe J R, Egger K N. Molecular Diversity of nifH Genes from Bacteria Associated with High Arctic Dwarf Shrubs. Microbial Ecology. 2006, 51: 516-525
33. Gotto J W, Taylor B F. N-fixation associated with decaying leaves of the red mangrove (Rhizophora mangle). Appl Environ Microbiol, 1976, 31: 781-783
34. Hamelin J, Iefromin N, Tarnawski S, Cuvelle S T, Aragno M. nifH gene diver sity in the bacterial, community associated with the rhizosphere of Molinia co erulea, an oligonitrophilic perennial grass. Environ Microbiol, 2002, 4 (8): 477 -481
35. Hicks B, Silvester W. Nitrogen fixation associated with New Zealand mangrov e (Avicennia marina Forsk). Appl Environ Microbiol, 1985, 49: 955-959
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