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Acta Microbiologica Sinica 2017, 57(4): 490-499 DOI: 10.13343/j.cnki.wsxb.

1 Acta Microbiologica Sinica 2017, 57(4): DOI: /j.cnki.wsxb Research Article 研究报告青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性沈硕 1,2,3* 摘要 : 16S rrna S rrna Bacillus Oceanobacillus Halomonas 关键词 : E N [1] [2] 168 km km 5856 km 2 4 [3] [4] [5 10] 3 基金项目 : (2014) * 通信作者 Tel Fax fjfzss@126.com 收稿日期 : 修回日期 : 网络出版日期 :

2 , 2017, 57(4) 491 DGGE 16 ARDRA [11] [12 13] 16S rrna 1 材料和方法 GPS ( 1) 45% ph C (1) ISP5 5 g 10 g L- 1 g K 2 HPO 4 1 g KNO 3 5 g MgCl 2 6H 2 O 50 g (FeSO 4 7H 2 O 0.2 g MnCl 2 4H 2 O 0.1 g ZnSO 4 7H 2 O 0.1 g 100 ml) 1 ml 12 g 1000 ml ph (2) 10 g 0.3 g KNO 3 2 g MgSO 4 7H 2 O 0.05 g K 2 HPO 4 2 g CaCO g FeSO 4 7H 2 O 10 mg NaCl 50 g 12 g 1000 ml ph (3) 表 1. 采样地点信息 Table 1. Information of sampling sites Sampling site Sample Longitude Latitude Altitude/m CHA01 Water E 95 12'58.7" N 36 46'8.6" 2681 CHA01 Mud E 95 12'58.7" N 36 46'8.6" 2681 CHA02 Mud E 95 16'29.2" N 36 47'4.2" 2678 CHA03 Water E 95 18'9.0" N 36 48'41.0" 2675 CHA03 Mud E 95 18'9.0" N 36 48'41.0" 2675 CHA04 Mud E 95 18'8.1" N 36 48'41.0" 2676 CHA05 Water E 95 18'6.6" N 36 48'42.1" 2676 CHA05 Mud E 95 18'6.6" N 36 48'42.1" 2676 CHA06 Water E 95 18'6.7" N 36 48'42.2" 2678

3 492 Shuo Shen Acta Microbiologica Sinica, 2017, 57(4) 10 g 0.3 g KNO 3 2 g MgSO 4 7H 2 O 0.05 g K 2 HPO 4 2 g CaCl 2 2H 2 O 1 g FeSO 4 7H 2 O 10 mg NaCl 50 g 12 g 1000 ml ph (4) CM 7.5 g 10 g 3 g MgSO 4 7H 2 O 20 g KCl 2 g FeSO 4 7H 2 O 0.05 g NaCl 30 g 12 g 1000 ml ph (5) ATCC213 MgSO 4 7H 2 O 10 g CaCl 2 2H 2 O 0.2 g KCl 5 g 2.5 g 10 g NaCl 40 g 12 g 1000 ml ph (6) M g 10 g 3 g MgSO 4 7H 2 O 20 g KCl 2 g FeSO 4 7H 2 O 0.36 g MnCl 2 4H 2 O 0.36 mg NaCl 30 g 12 g 900 ml ph C Na 2 CO ml (7) APA NaCl 50 g KH 2 PO 4 2 g MgSO 4 7H 2 O 1 g 5 g 5 g 1 g 12 g 800 ml ph C 5% Na 2 CO ml (8) RM NaCl 50 g MgSO g 3.0 g KCl 2.0 g CaCl g 10.0 g 2.0 g 12 g 1000 ml ph C 30 min 0.22 µm 30 ml 3 ml g 90 ml 30 min [14] μl 28 C 37 C 4 C [14] S rrna 16S rdna ( ) DNA PCR 10 Buffer 2.5 μl DNA 1 μl Taq (5 U /μl) 0.2 μl dntps (2.5 mmol/μl) 2 μl F27 (5 -AGAGTTTGATCCTGGCTCAG G-3 ) P1541(5 -AAGGAGGTGGTGATCCA GCCGCA-3 ) (10 pmol/μl) 1 μl 25 μl 94 C 5 min 94 C 30 s 55 C 30 s 72 C 80 s C 10 min PCR S rrna EzTaxon ( 16S rrna Mega 5.0 actamicro@im.ac.cn

4 , 2017, 57(4) 493 [15] S rdna 97% (OTU) Shannon Margalef Shannon Berger-Parker [16] 2 结果和分析 E (ATCC213 ) 190 D (CM ) 91 H (RM ) 88 ( 2) S rrna 16S rrna 98.73% % ( 1 2) 4 Bacillaceae 16S rrna S95 Bacillus solimangrovi (GH2-4) 16S rrna 96.79% S115 Ornithinibacillus contaminans (CCUG 53201) 16S rrna 97.98% S165 Bacillus alcalophilus (DSM 485) 16S rrna 97.30% S181 Ornithinibacillus contaminans (CCUG 53201) 16S rrna 95.34% 4 83, 16S rrna 83 Firmicutes Proteobacteria Actinobacteria ( 1 2) 表 2. 不同的察尔汗盐湖样品和培养基中分离中度嗜盐菌菌株数量 Table 2. The number of the moderate halophiles isolated from different Qrhan Salt Lake samples and culture media Moderate halophiles isolated from different samples Moderate halophiles isolated from different culture media Sample No. Sample Strain number Media No. Media Strain number 1 Water A 2 2 Mud 38 2 B 0 3 Mud 8 3 C 0 4 Water 43 4 D 91 5 Mud 21 5 E Mud 6 6 F 29 7 Water 66 7 G 21 8 Mud 33 8 H 88 9 Water 11 Total

5 494 Shuo Shen Acta Microbiologica Sinica, 2017, 57(4) 图 1. 察尔汗盐湖可培养细菌 16S rrna 基因序列 Firmicutes 和 Actinobacteria 门的 NJ 系统发育树 Figure 1. Neighbor-Joining tree showing the phylogenetic relationships of culturable bacteria from Qrhan Salt Lake based on 16S rrna gene sequences for Firmicutes and Actinobacteria. The tree rooted was constructed by Neighbor-Joining method with bootstrap values calculated from 1000 resampling. The numbers at each node that indicated the percentage of bootstrap supporting. The numbers in the brackets after each strain name are accession numbers of 16S rrna gene sequences in GenBank. Bar 0.02 at the bottom is the sequence digergence.

6 , 2017, 57(4) 495 图 2. 察尔汗盐湖可培养细菌 16S rrna 基因序列 Proteobacteria 门的 NJ 系统发育树 Figure 2. Neighbor-Joining tree showing the phylogenetic relationships of culturable bacteria from Qrhan Salt Lake based on 16S rrna gene sequences for Proteobacteria. The tree rooted was constructed by Neighbor-Joining method with bootstrap values calculated from 1000 resampling. The numbers at each node that indicated the percentage of bootstrap supporting. The numbers in the brackets after each strain name are accession numbers of 16S rrna gene sequences in GenBank. Bar at the bottom is the sequence digergence. Firmicutes Bacillus Gracilibacillus Paraliobacillus Halobacillus Bacillus_g26 Bacillus_g6 Virgibacillus Ornithinibacillus Oceanobacillus Terribacillus Staphylococcus Fictibacillus12 Actinobacteria Streptomyces Kocuria Micrococcus3 ( 1 3) Proteobacteria Halomonas ( 2 3) Firmicutes Bacillus Oceanobacillus Proteobacteria Halomonas Firmicutes Bacillus 10 ( 3) ( 4) Shnnon-wiener Margalef Shannon Eveness Berger-Parker dominance Index 表 3. 察尔汗盐湖可培养嗜盐菌菌株在属水平上的相对丰度 Table 3. Relative abundance of culturable bacteria in the genus level in Qrhan Salt Lake Phylum Genus Water sample Strain number Firmicutes Staphylococcus 1 2 Fictibacillus 0 1 Bacillus 1 0 Terribacillus 2 0 Oceanobacillus 15 2 Ornithinibacillus 2 0 Virgibacillus 2 0 Bacillus_ g6 1 0 Bacillus_ g Halobacillus 2 0 Paraliobacillus 1 0 Gracilibacillus 2 0 Bacillus Proteobacteria Halomonas 10 5 Actinobacteria Streptomyces 0 1 Kocuria 0 1 Micrococcus 1 0 Mud sample

7 496 Shuo Shen Acta Microbiologica Sinica, 2017, 57(4) Sample Table 4. Number of isolates 表 4. 主要中度嗜盐菌多样性指数值 The Indexes of biodiversity of main moderate halophilic bacteria Shannon-wiener Index (H ) Margalef Index (D Mg ) Shannon Evenness (E) Water part Mud part Total Berger-Parker dominance Index (d) 3 讨论 [17] 86 [9] [18 20] [16,21] ARDRA 60 [22 23] 1 (Marinobacter) [24] [25 26] 83 4 Bacillaceae 16S rrna Firmicutes 3 Firmicutes Bacillus Oceanobacillus Proteobacteria Halomonas 1 Firmicutes Bacillus Bacillus actamicro@im.ac.cn

8 , 2017, 57(4) 497 DGGE ARDRA [27 30] Bacillus 4 Bacillaceae Bacillus [31 33] Bacillus 4 [1],,,,,.., 2008, 15(5): [2],.., 2010, (19): 215. [3] Zhang ZG, Li BX. Salt karst research of Qarhan Salt Lake in Qinghai province. Journal of Salt Lake Research, 2008, 16(3): (in Chinese),.., 2008, 16(3): [4] Li CB, Zhang XC. Status quo of development of potassium resource in Cha-er-han Salt Lake in Qinghai. Modern Mining, 2009, (2): (in Chinese),.., 2009, (2): [5] Lim JM, Jeon CO, Kim CJ. Bacillus taeanensis sp. nov., a halophilic Gram-positive bacterium from a solar saltern in Korea. International Journal of Systematic and Evolutionary Microbiology, 2006, 56(12): [6] Usami R, Echigo A, Fukushima T, Mizuki T, Yoshida Y, Kamekura M. Alkalibacillus silvisoli sp. nov., an alkaliphilic moderate halophile isolated from non-saline forest soil in Japan. International Journal of Systematic and Evolutionary Microbiology, 2007, 57(4): [7] Liebgott PP, Joseph M, Fardeau ML, Cayol JL, Falsen E, Chamkh F, Qatibi AI, Labat M. Clostridiisalibacter paucivorans gen. nov., sp. nov., a novel moderately halophilic bacterium isolated from olive mill wastewater. International Journal of Systematic and Evolutionary Microbiology, 2008, 58(1): [8] Antunes A, Rainey FA, Wanner G, Taborda M, Pätzold J, Nobre MF, da Costa MS, Huber R. A New lineage of halophilic, wall-less, contractile bacteria from a brine-filled deep of the Red Sea. Journal of Bacteriology, 2008, 190(10): [9] Borsodi AK, Márialigeti K, Szabó G, Palatinszky M, Pollák B, Kéki Z, Kovács AL, Schumann P, Tóth EM. Bacillus aurantiacus sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from Hungarian soda lakes. International Journal of Systematic and Evolutionary Microbiology, 2008, 58(4): [10].., [11] Chai LH, Wang T, Li QY, Cui XL, Peng Q, Xu LH, Jiang CL. Assessment of the bacterial community in the two adjacent lakes, Qinghai, by DGGE. Journal of Biology, 2003, 20(1): 13 14, 19. (in Chinese),,,,,,. DGGE., 2003, 20(1): 13 14, 19. [12] Chai LH, Wang T, Cui XL, Peng Q, Xu LH, Jiang CL. A primary study of 16 strains in Keke Salt Lake by ARDRA and phylogenetic analysis. Journal of Yunnan University (Natural Sciences Edition), 2003, 25(6): (in Chinese),,,,,. 16 ARDRA. ( ), 2003, 25(6): [13] Jiang Y, Li WJ, Xu P, Tang SK, Xu LH, Zhang YQ, Zhang YG, Zhang LP. Study on diversity of actinomycetes under salt

9 498 Shuo Shen Acta Microbiologica Sinica, 2017, 57(4) and alkaline environments. Acta Microbiologica Sinica, 2006, 46(2): (in Chinese),,,,,,,.., 2006, 46(2): [14] Shen S, Wang J. Isolation, petrifaction and inhibitory activity against plant pathogenic fungi of halophilic strains from Qinghai Salt Lake. Guangdong Agricultural Sciences, 2013, 40(1): 79 81, 88. (in Chinese),.., 2013, 40(1): 79 81, 88. [15] Sun HM, Wei YZ, Fang XM, Yu LY, Zhang YQ. Diversity of endophytic bacteria isolated from Huperzia serrata. Acta Microbiologica Sinica, 2016, 56(4): (in Chinese),,,,.., 2016, 56(4): [16] Xu XW, Wu M, Dilbar T, Gulibahaer A. Halophilic archaea diversity of Aibi Lake and Yiwu Lake in Xinjiang. Biodiversity Science, 2006, 14(4): (in Chinese),,,.., 2006, 14(4): [17] Zhao W. The current research status and prospectives of organisms in Chinese Saline Lakes. Journal of Dalian Fisheries University, 2009, 24(5): (in Chinese).., 2009, 24(5): [18] Liu HQ, Che HJ. The numerical disposal of halophilic microbiology from Dabancheng Saline Lake in Xinjiang. Journal of Xinjiang Normal University (Natural Sciences Edition), 2006, 25(3): , 111. (in Chinese),.. ( ), 2006, 25(3): , 111. [19] Liu HQ, Zhao CM, Huang RH. The classification research of moderately halophilic bacteria from Dabancheng Saline Lake in Xinjiang. Journal of Xinjiang Normal University (Natural Sciences Edition), 2007, 26(3): , 159. (in Chinese),,.. ( ), 2007, 26(3): , 159. [20] Liu HQ, Lv JJ. The 16S rdna sequence research of moderately halophilic bacteria from Dabancheng Saline Lake in Xinjiang. Journal of Xinjiang Normal University (Natural Sciences Edition), 2008, 27(4): (in Chinese),. 16S rdna. ( ), 2008, 27(4): [21].., [22] Pan HL, Zhou C, Wang HL, Xue YF, Ma YH. Diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China. Acta Microbiologica Sinica, 2006, 46(1): 1 6. (in Chinese),,,,.., 2006, 46(1): 1 6. [23].., [24].., [25].., [26] Zhou XH, Wang Y, Wu M. Isolation anti exopolysaeeharids screening of halophiles from Zhoushan Islands. Journal of Zhejiang University (Science Edition), 2007, 34(3): (in Chinese),,.. ( ), 2007, 34(3): [27] Fan HP, Xue YF, Zeng Y, Zhou PJ, Ma YH. Archaeal diversity of Zabuye Lake in Tibet analyzed by culture-independent approach. Acta Microbiologica Sinica, 2003, 43(4): (in Chinese),,,,.., 2003, 43(4): [28] Chen M, Zhao LP. Biodiversity of bacterial isolates on three different media from coking wastewater treatment system. Acta Microbiologica Sinica, 2003, 43(3): (in Chinese),.., 2003, 43(3): [29] Zhao S, Zhou N, Zhao ZY, Zhang K, Tian CY. Endophytic bacterial diversity and dynamics in root of Salicornia europaea estimated via high throughput sequencing. Acta Microbiologica Sinica, 2016, 56(6): (in Chinese),,,,.., 2016, 56(6): [30] Xia WW, Jia ZJ. Comparative analysis of soil microbial communities by pyrosequencing and DGGE. Acta Microbiologica Sinica, 2014, 54(12): (in Chinese),. DGGE., 2014, 54(12): [31] Zhang H, Li PJ, Hu XM, Wang X, Verkhozina VA. Study on bioremediation of oil-contaminated surface water by using immobilized Bacillus sp. China Water & Wastewater, 2007, actamicro@im.ac.cn

10 , 2017, 57(4) (7): (in Chinese),,,, Verkhozina VA.., 2007, 23(7): [32] Jin ZX, Wu WQ, Zhang Z. The starch of dioscorea zingibernisis effects to α-amylase activity in Bacillus subtilis. Journal of Microbiology, 2004, 24(4): 23 24, 45. (in Chinese),,. α-., 2004, 24(4): 23 24, 45. [33] Liu XH. Applied research and development of Bacillus. Journal of Shangqiu Teachers College, 2005, 21(5): (in Chinese).., 2005, 21(5): Community structure and diversity of culturable moderate halophilic bacteria isolated from Qrhan Salt Lake on Qinghai-Tibet Plateau Shuo Shen 1,2,3* 1 Academy of agriculture and forestry sciences, Qinghai University, Xining , Qinghai Province, China 2 State key laboratory of plateau ecology and agriculture, Qinghai university, Xining , Qinghai Province, China 3 The Qinghai-Tibet Plateau Biotechnology Key Laboratory of Ministry of Education, Qinghai University, Xining , Qinghai Province, China Abstract: [Objective] I studied the community structure and diversity of culturable moderate halophilic bacteria isolated from Qrhan Salt Lake. [Methods] I isolated and cultured the moderate halophilic bacteria on different selective media. After the 16S rrna gene sequences was amplified and measured, I constructed the phylogenic tree, analyzed the community structure and calculated the diversity indexes according to the 16S rrna gene information. [Results] A total of 421 moderate halophilic bacteria were isolated from water and mud samples in Qrhan Salt Lake. The 16S rrna gene information showed that 4 potential novel species belonged to the family Bacillaceae. Eighty-three model strains belonged to 3 phylurms 6 families 16 genus. Among them, Bacillus sp., Oceanobacillus sp. and Halomonas sp. were dominant species. Diversity analysis showed that the diversity of strains isolated from water sample was higher than that from mud sample, but the dominance degree of strains isolated from mud sample was higher than that from water sample. [Conclusion] The genetic diversity of moderate halophilic bacteria isolated from Qrhan Salt Lake was abundant. Also, there were dominant and novel species of culturable moderate halophilic bacteria in this lake. Keywords: Qrhan Salt Lake, moderate halophilic bacteria, community structure, diversity ( 本文责编 : 张晓丽 ) Supported by The Western Light Cultivation Plan of Chinese Academy of Sciences (2014) and by the Foundation of Key Laboratory of the Qinghai-Tibet Plateau Biotechnology, Ministry of Education * Corresponding author. Tel: ; Fax: ; fjfzss@126.com Received: 7 June 2016; Revised: 17 October 2016; Published online: 4 November 2016

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇