微生物学通报 MAY 20, 2008, 35(5): 737~742 Microbiology 2008 by Institute of Microbiology, CAS 研究报告连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究 * 何敏艳邹正中蔡林王革娇 ( 4

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1 微生物学通报 MAY 20, 2008, 35(5): 737~742 Microbiology 2008 by Institute of Microbiology, CAS 研究报告连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究 * 何敏艳邹正中蔡林王革娇 ( ) 摘要 : 盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究, 结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性采用培养法从两地盐土中共分离到 17 株嗜盐细菌, 其中 Halomonas 为两地盐土共有的嗜盐细菌, 而 Halobacillus 和 Pontibacillus 仅在三圩盐土中发现通过非培养的 16S rdna 基因文库法从两地盐土中发现了 13 种嗜盐古菌, 台北盐土有 Halobacterium 和 Haloplanus, 三圩盐土有 Halobacterium, Natronobacterium, Halogeometricum 和 Haloarcula 10 个嗜盐古菌的 16S rdna 和 GenBank 已知序列的同源性为 92%~97%, 可能为这些属中的新种该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础关键词 : 盐田土, 嗜盐细菌, 嗜盐古菌, 微生物多样性 Polymorphic Halophilic Bacteria and Haloarchaea from Lianyungang Taibei and Yancheng Sanwei Salt Field Soils HE Min-Yan ZOU Zheng-Zhong CAI Lin WANG Ge-Jiao * (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan ) Abstract: Halophilic microorganisms play important rules in salt field ecosystem and salt production. In this study, halophilic bacteria and haloarchaea from soils of Lianyungang Taibei and Yancheng Sanwei salt fields were analyzed. The halophilic bacterial and haloarchaeal types from both the soils were similar, but each soil had its distinctive species. A total of 17 halophilic bacteria were identified, among them, Halomonas was found from both the soils, while Pontibacillus and Halobacillus were isolated from Sanwei salt field only. Using uncultured 16S rrna gene library technology, 13 haloarchaeal soil 16S rrna genes were identified from both the saline soils. Halobacterium and Haloplanus were found from Taibei salt field, while Halobacterium, Natronobacterium, Halogeometricum and Haloarcula were identified from Sanwei salt field. Ten haloarchaeal 16S rrna gene sequences showed 92%~97% identities with the GenBank sequences that appear to represent novel soil haloarchaeal species. This study provides important information that is useful for further investigation and application of halophiles of saline soil fields. Keywords: Salt field soil, Halophilic bacteria, Haloarchaea, Microbial diversity 基金项目 : (No ) ; (No. 2005DKA ) * 通讯作者 :Tel: ; : gejiao@mail.hzau.edu.cn 收稿日期 : ; 接受日期 :

2 738 微生物学通报 2008, Vol.35, No.5, [1] ;, [2], NaCl 3 (0.2 mol/l~0.5 mol/l) (0.5 mol/l~ 3.0 mol/l) (2.5 mol/l~5.2 mol/l), [3], ( ) [4,5],,, [2], Walsh [6],,,,, 1 材料和方法 1.1 样品采集与处理 , 30 km 10 ( 200 m), 15 cm, 2 mm,, 4, 20 DNA 1.2 土样理化性质的测定 [7] ; Na + K + Mg 2+ ( ) ; ph [8] 1.3 嗜盐菌的分离与保藏 HGM [9] CM [10], 10 g 90 ml, min,, 嗜盐菌耐盐性的测定 NaCl HGM NaCl 0 mol/l~5.0 mol/l, 0.5 mol/l, 37, 200 r/min 24 h (OD 600 ) 1.5 嗜盐细菌 16S rrna 基因的鉴定 PCR 16S rdna 50 µl, min, 5 min, r/min 5 min, 2 µl 16S rdna PCR PCR 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) 1492R(5 - GGYTACCTTGTTACGACTT-3 ) [11] PCR 94 5 min; 94 1 min, 49 1 min, 72 2 min, 35 ; min PCR HaeIII ( PCR-RFLP ), PCR 1.6 非培养法构建嗜盐古菌 16S rrna 基因文库分析 DNA [12], DNA PCR 16S rrna A27F (5 -TCCGGT TGATCCTGCCGGAG-3 ) A1540R (5 -AGGAGG TGATCCAGCCGCAG-3 ) [13], PCR PCR pgem-t (Promega, USA), E. coli DH5α, Amp X-Gal IPTG LB, h 100, 16S rdna PCR,

3 : 嗜盐细菌和古菌的多样性分析 16S rdna Blastn GenBank ( ncbi. nlm.nih.gov/blast), ClustalW, MEGA3.1 ( software.net) 1.8 核苷酸序列注册号 16S rdna NCBI GenBank, EF533960~EF EF533946~ EF 结果 2.1 土壤理化性质测定 Na + K + Mg 2+ ph, Na + K + Mg ( 1) 2.2 嗜盐细菌的分离鉴定 120,, 30, PCR-RFLP 17, 4, 13 ( 1) NaCl 0.5 mol/l~4.5 mol/l ; NaCl 0.5 mol/l 1.0 mol/l 1.5 mol/l 2.0 mol/l ( 2) 16S rdna Table 1 表 1 台北和三圩盐场盐田土理化性质测定结果 Results of physical and chemical properties of the soil of Taibei and Sanwei salt fields Sampling sites Texture of soil ph Na + (g/kg) K + (g/kg) Mg 2+ (mg/kg) (%) Organic carbon Taibei salt field Sandy soil Sanwei salt field Clay Table 2 表 2 台北盐田和三圩盐田土嗜盐菌部分特征 Partial characters of the halophiles of Taibei (L) and Sanwei (Y) saline soils Strain GenBank The highest identity with GenBank bacterial species Sequence identity (%) NaCl Range of NaCl concentration (mol/l) NaCl Optimum NaCl concentration (mol/l) Y12 Halomonas koreensis (AY382579) Y13 Halobacillus trueperi (DQ157162) Y22 Halomonas ventosae (DQ316071) Y23 Halomonas taeanensis (AY671975) Y24 Halobacillus sp. (AY121438) Y31 Halomonas sp. (AB305247) Y32 Pontibacillus sp. (DQ448765) Y33 Halomonas variabilis (AY505527) Y34 Pontibacillus marinus (AY603977) Y35 Halobacillus locisalis (AY190534) Y38 Bacillus halophilus (AB021188) Y71 Halomonas alimentaria (AF211860) Y72 Halomonas ventosae (DQ316071) L10 Halomonas sp. (AM229323) L11 Halomonas sp. (AM229323) L18 Halomonas denitrificans (AM ) L24 Halomonas alkantarctica (AJ564880)

740 微生物学通报 2008, Vol.35, No.5 图 1 17 种嗜盐细菌的 16S rdna-haeiii 酶切琼脂糖凝胶电泳图 (2 ) Fig.

Halomonas (11 ), Pontibacillus(2 ), Halobacillus(4 ) 3, 97 ~100 Halomonas, Halobacillus Pontibacillus 2.

98 ; LA52 Haloplanus natans 97 ; YA46 Haloarchaeon Nie7-1 97 ; YA6 Natronobacteriums pharaonis 92 ; YA8 Haloarcula marismortui 96 ; LA9 LA11 LA54 YA9 YA66 Halobacterium

4 740 微生物学通报 2008, Vol.35, No.5 图 1 17 种嗜盐细菌的 16S rdna-haeiii 酶切琼脂糖凝胶电泳图 (2 ) Fig. 1 An agarose gel electrophoresis (2 ) showing HaeIII digested fragments of the 16S rdnas from the 17 halophilic bacteria M: M: Molecular marker 2, 17 (Group ) (L) (Y) Halomonas (11 ), Pontibacillus(2 ), Halobacillus(4 ) 3, 97 ~100 Halomonas, Halobacillus Pontibacillus 2.3 嗜盐古菌的鉴定 16S rdna S rdna, S rdna Halobacterium Haloplanus Natronobacterium Halogeometricum Haloarcula, LA15 YA1 YA39 YA32 Halobacterium Saccharovorum, ; LA52 Haloplanus natans 97 ; YA46 Haloarchaeon Nie ; YA6 Natronobacteriums pharaonis 92 ; YA8 Haloarcula marismortui 96 ; LA9 LA11 LA54 YA9 YA66 Halobacterium Saccharovorum ,, 图 3 两地盐土嗜盐古菌 16S rdna 系统发育树 Fig. 3 16S rdna phylogenetic tree of the haloarchaeal bacteria from Taibei (L) and Sanwei (Y) saline soils 图 2 两地盐土嗜盐细菌 16S rdna 系统发育树 Fig. 2 16S rdna phylogenetic tree of the halophilic bacteria from Taibei (L) and Sanwei (Y) saline soils 3 讨论 118 o o 48, o 07, ,

5 : ,,, 14,,,,,,,,, (Halomonas) (Halobacillus), Pontibacillus 4 Halomonas Halobacillus Pontibacillus, ( ), Halorubrum Natronococcus Natronorubrum Haloterrigena Halorhabdus, [14] Natronococcus Natronomonas Natronorubrum Natrinema Natronobacterium Halorubrum Haloterrigena Halorhabdus 8,, 16S rdna, 16S rdna 5 (Natronobacterium) (Halobacterium) (Haloplanus) (Halogeometricum) (Haloarcula) Halobacterium, Haloplanus, Natronobacterium Halogeometricum Haloarcula Walsh [6] Na + K + Mg 2+,,,, 6, (Natronococcus) (Halorubrum) (Natronobacterium) (Natrialba) (Natronorubrum) (Natronomonas) (ph 7.95) 1 YA6 (Natronobacterium), ph (ph 8.35), ph, 16S rrna, 13 3 Halobacterium Saccharovorum 98 ~99, 10 GenBank 16S rrna 92 ~97,,,, 参考文献 [1] Paterek JR, Smith PH. Isolation and characterization of a halophilic methanogen from Great Salt Lake. Applied and Environmental Microbiology, 1985, 50(4): [2],,,.., 2004, 25(1): [3] Vreeland RH. Mechanisms of halotolerance on microorganisms. CRC Critical Reviews in Microbiology, 1987, 14(4): [4],,,.., 2006, 46(2): [5],,,. alkalip., 2006, 46(1): 1 6. [6] Walsh DA, Papke RT, Doolittle WF. Archaeal diversity

742 微生物学通报 2008, Vol.35, No.5 along a soil salinity gradient prone to disturbance. Environmental Microbiology, 2005, 7(10): 1655 1666. [7],,.

Aerobic metabolism of 4-hydroxybenzoic acid in archaea via an unusual pathway involving an intramolecµlar migration.

Can J Microbiol, 1969, 15: 1157 1165. [11] Wilson KH, Blitchington RB, Greene RC.

DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 1996, 62(2): 316 322. [13],,,.., 2006, 14(4): 359 362. [14],,,.

6 742 微生物学通报 2008, Vol.35, No.5 along a soil salinity gradient prone to disturbance. Environmental Microbiology, 2005, 7(10): [7],,.., 2005, 2: [8],,. ISO10390: 2005 ph., 2006, 1: [9] Fairley DJ, Boyd DR, Sharma ND, et al. Aerobic metabolism of 4-hydroxybenzoic acid in archaea via an unusual pathway involving an intramolecµlar migration. Appl Environ Microbiol, 2002, 68(12): [10] Gochnauer MB, Kushner DJ. Growth and nutrition of extremely halophilic bacteria. Can J Microbiol, 1969, 15: [11] Wilson KH, Blitchington RB, Greene RC. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol, 1990, 28: [12] Zhou JZ, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 1996, 62(2): [13],,,.., 2006, 14(4): [14],,,.., 2003, 43(4): 编辑部公告 2008 年中科院微生物所期刊联合编辑部联合征订全面启动! ( 4 ), 55.00, 660 ISSN ; CODEN WSHPA ; BM67 ( 25 ), 65.00, 780 ISSN ; CODEN SGXUED 82-13; BM5608 ( 20 ), 48.00, 576 ISSN ; CODEN WSWPDI 2-817; BM413 ( 15 ), 80, 480 ISSN /Q, CODEN JXUUAE 2-499; Q723 (100101) B401 ; (010) ; hanl@im.ac.cn

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Microsoft Word - 崔宝臣.doc 微生物学通报 DEC 20, 2009, 36(12): 1871~1875 Microbiology tongbao@im.ac.cn 2009 by Institute of Microbiology, CAS 简报正交实验优选光合细菌混合菌群产氢的最佳条件 * 崔宝臣张国欣侯博邵姝媛 ( 163318) 摘要 : 对光合细菌混合菌群产氢影响因子进行了实验研究通过单因素实验和正交实验, 系统考察了碳源

微生物学通报 MAY 20, 2008, 35(5): 737~742 Microbiology 2008 by Institute of Microbiology, CAS 研究报告 连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究 * 何敏艳邹正中蔡林王革娇 ( 4

微生物学通报 MAY 20, 2008, 35(5): 737~742 Microbiology 2008 by Institute of Microbiology, CAS 研究报告连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究 * 何敏艳邹正中蔡林王革娇 ( 4