收稿日期: 2008-12-05
修回日期: 2009-02-06
网络出版日期: 2011-10-10
基金资助
中国科学院知识创新项目(KSCX2?SW?132); 国家自然科学基金(40776069、40676091); 国家生态系统研究网络三亚站
数据库与信息系统, 中科院生态系统研究网络台站研究基金; 海洋公益性行业科研专项经费项目(200705026); 广东省科
技计划项目(2004B60302004, NO.2005B60301032); 重大基础研究前期研究专项(2005CCA04800); 广东省海洋药物重
点实验室开放课题; 中国科学院南海海洋研究所LMM-LAMB-LMB联合开放课题。
Isolation and characterization of a N2-Fixing Bacterium from the mangrove rhizosphere and study on its nitrogen-fixing ability
Received date: 2008-12-05
Revised date: 2009-02-06
Online published: 2011-10-10
Supported by
中国科学院知识创新项目(KSCX2?SW?132); 国家自然科学基金(40776069、40676091); 国家生态系统研究网络三亚站
数据库与信息系统, 中科院生态系统研究网络台站研究基金; 海洋公益性行业科研专项经费项目(200705026); 广东省科
技计划项目(2004B60302004, NO.2005B60301032); 重大基础研究前期研究专项(2005CCA04800); 广东省海洋药物重
点实验室开放课题; 中国科学院南海海洋研究所LMM-LAMB-LMB联合开放课题。
采用选择性无氮培养基从红树林根际土壤中分离出一株具有高效固氮活性的固氮细菌, 通过形态学, 生化鉴定, G+C摩尔分数含量和16S rRNA以及固氮基因nif H的序列分析, 初步鉴定为短小芽孢杆菌Bacillus pumilus。其特征是该菌为革兰氏阳性, 直杆状, 固体培养基上形成圆形白色菌落(直径3—4mm), 与短小芽孢杆菌标准菌株相比较, 它们在碳源利用, 水解以及生长温度盐度等方面具相似程度很高, G+C 摩尔分数含量为44.6%, 以16S rRNA为基础构建的系统进化树分析其与短小芽孢杆菌B. pumilus B402的进化距离最近, 相似性为99.6%, 利用乙炔还原法对其固氮活性进行测定, 具有较高的固氮活性, 为156.32nmol C2H4•H?1•mL?1。
凌娟,董俊德,张燕英,杨志浩,王友绍 . 一株红树林根际固氮菌的分离、鉴定以及固氮活性测定[J]. 热带海洋学报, 2010 , 29(5) : 149 -153 . DOI: 10.11978/j.issn.1009-5470.2010.05.149
A bacterial strain H9 capable of nitrogen fixation was isolated from the mangrove rhizosphere using N-free medium. It was identified as Bacillus pumilus by type description, physiological and biochemical test, G+C Mole fraction (%), 16S rRNA sequence analysis and the nif H sequence analysis. It was a gram-positive, straight-rodded bacterium, which forms white colony with diameter of 3-4 mm on the solid agar medium. Compared with the standard strain of the species B. pumilus, there were high similarity in the carbon source utility, hydrolysis and the optimal growth temperature and salinity. The G+C content of the strain DNA was 44.6%. A phylogenetic tree was constructed by comparing validly published 16S rRNA sequences of the related strains in the Genebank, using the neighbor-joining method. It showed that the strain H9 and the strain B. pumilus B402 have the highest similarity, which is 99.6%. The measurement of nitrogen-fixing ability by acetylene reduction was 156.32 nmol C2H4•H?1•mL?1, indicating its high nitrogen-fixing ability.
Key words： mangrove; nitrogen-fixing bacterium; 16S rRNA; nif H
[1] 林鹏. 海洋高等植物生态学[M]. 北京: 科学出版社, 2006: 76-80.
[2] 黎遗业. 广西沿海红树林的生态保护政策 [J]. 资源开发与市场, 2008, 24(3): 267-269.
[3] ALONGI D M, CHRISTOFFERSEN P, TIRENDI F, et al. The influence of forest type on microbial-nutrient relationships in tropical mangrove sediments[J]. J Exp Mar Biol Ecol, 1993, 11: 201-203.
[4] SENGUPTA A, CHAUDHURI S. Ecology of heterotrophic dinitrogen fixation in the rhizosphere of mangrove plant community at the Ganges river estuary in India[J]. Oecologia, 1991, 87: 560-564.
[5] HOLGUIN G, GUZMAN M A, BASHAN Y, et al. Two new nitrogen-fixing bacteria from the rhizosphere of mangrove trees: their isolation, identification and in virtro interaction with rhizosphere of Staphylococcus sp.[J]. FEMS Microbial Ecol, 1992, 101: 207-216.
[6] 韩威威, 沈世华, 荆玉祥. 生物固氮中的蛋白质组学[J]. 农业生物技术学报, 2004, 12(4): 464-494.
[7] HOLGUIN G, BASHAN Y. Nitrogen fixation by Azospirillum brasilense Cd is promoted when co-cultured with a mangrove rhizosphere bacterium staphylococcus sp[J]. Soil Biol Biochem, 1996, 28(12): 1651-1660.
[8] TOLEDO G, BASHAN Y. In vitro colonization and increase in nitrogen fixation of seedling roots of black mangrove inoculated by a filamentous cyanobacteria[J]. Can J Microbiol, l995, 41(11): 1012-1020.
[9] ADRIANA R, GINA H, YOAV B, et al. Synergism between Phyllobacterium sp. (N2-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere[J]. FEMS Microbiol Ecol, 2001, 35(2): 181-187.
[10] 林鹏. 红树林研究论文集: 第l集[G]. 厦门: 厦门大学出版, 1990.
[11] 林鹏. 红树林研究论文集: 第2集[G]. 厦门: 厦门大学出版, 1993.
[12] 林鹏. 红树林研究论文集: 第3集[G]. 厦门: 厦门大学出版, 1999.
[13] 林鹏. 红树林研究论文集: 第4集[G]. 厦门: 厦门大学出版, 2000.
[14] 尤崇杓, 姜涌明, 宋鸿雨. 生物固氮[M]. 北京: 科学出版社, 1987, 224-245.
[15] 曾定. 固氮生物学[M]. 厦门: 厦门大学出版社, l987, 260-263.
[16] 林敏, 方宜钧, 程红梅, 等. 固氮细菌氮调系统组成及调控机制. 农业生物技术学报[J], 1995, 3(1): 28-33.
[17] 章生卫, 章金鸿, 罗海鲲. 利用植物生长促进菌(PGPB)恢复红树林[J]. 广州环境科学, 2002, 17(2): 1-4.
[18] RACIKUMAR S, KATHIRESAN K, THADEDUS S, et al. Nitrogen-fixing azotobacters from mangrove habitat and their utility as marine biofertilizers [J]. J Exp Mar Bio, 312 (2004): 5-17.
[19] 梁静娟, 王松柏, 庞宗文, 等. 海洋细菌Bacillus pumilus PLM4产抗肿瘤多糖的发酵条件优化研究[J]. 广西农业生物科学, 2006, 25(3): 256-260.
[20] 耿彦生, 李涛. 短小芽孢杆菌E601抗电离辐射研究[J]. 中华流行病学杂志, 1999, 20(6): 334-337.
[21] CHANG B V, CHANG I T, YUAN S Y. Biodegradation of phenanthrene and pyrene from mangrove sediment in subtropical Taiwan[J]. J environ sci health, 2008, 43(3): 233-238.
[22] 王逸群, 郑金贵, 陈文列, 等. 稻内生固氮菌的分离及鹑鸡肠球菌在水稻根中的分布[J]. 热带亚热带植物学报, 2005, 13(4): 296-302.
[23] 赵斌, 何绍江. 微生物学实验[M]. 北京: 科学出版社, 2002, 5(2): 254-255.
[24] 谢光辉. 长江流域水稻根际芽胞杆菌属固氮菌株的分离与鉴定[J]. 微生物学报, 1998, 38(6): 480-483.
[25] XIE G H, CAI M Y, TAO G C, et al. Cultivable Heterotrophic N2-Fixing Bacterial Diversity in Rice Field along Yangtzs River Plain[J]. Biol Fert Soils, 2003, 37: 29-38.
[26] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001: 330-336.
[27] MARMUR J. Thermal renaturation of deoxyribonucleic acids[J]. J Mol Biol, 1961, 3: 585-589.
[28] 田新朋. 云南黑井盐矿可培养极端嗜盐古菌的分离及系统学研究[D]. 昆明: 云南大学硕士论文, 2006.
[29] 何延静, 刘海明, 胡洪波, 等. 一株拮抗辣椒疫霉的假 单胞菌的分离与鉴定[J]. 微生物学报，2006, 46(4), 516-521.
[30] ORSINI M, RONMANO-SPICA V. A microwave-based method for nucleic acid isloation from environmental samples[J]. Lett Appl Microbiol Lett, 2001, 33: 17-20.
[31] 徐平, 李文均, 徐丽华. 微波法快速提取放线菌基因组DNA[J]. 微生物学通报, 2003, 30(4): 82-84.
[32] WEISBURG W, BARNS S, PELLETIER D. 16S ribosomal DNA amplification for phylogenetic study[J].J BACTERIOL, 1991, 173(2): 697-703.
[33] POLY F, RANJARD L, NAZARET S, et al. Comparison of nifH gene pools between soils and between soil microenvironments of contrasting properties[J]. APPL ENVIRON MICROB, 2001(67): 225-2267.
[34] SAITOU N, NEI M. The neighbor-joining method: A new method for reconstructing phylogenetic tree[J]. Mol Biol Evol, 1987, 4: 406-425.
[35] KIMURA M. A simple method for estimating evolutionary rates of base substitutions nucleotide sequence[J]. J Mol Evol, 1980, 16: 111-120.
[36] KIMURA M. The neutral theory of molecular evolution[M]. New York: Cambridge University Press, 1983.
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