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1 46 5 Vol.46, No OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2015 FA08 * 1, ( ; ; ), FA08,, 16S rdna, FA08 (Bacillus licheniformis, GenBank : KM892857) FA08 ph, FA08, ph 3 ph 10,, C, 70%, FA08,, FA08, ; ; ; ; Q doi: /hyhz ,,,,, (Vanittanakom et al, 1986; Burgess et al, 1993; Barsby et al, 2001; Vaseeharan et al, 2003;, 2011;, 2011;, 2012;, 2012;, 2012);,,,, LHB02 (Vibrio harveyi) (Vibrio alginolyticus) (Vibrio anguillarum) (, 2011); ( ) (, 2008) (Tamehiro et al, 2002; Liu et al, 2010),,,,, mrna (, 2007);,,,, (, 2014);,, (, 2004);,,,, (, 2006), *, 2013Y0063 ;, 13GZP002NF08,, fangweidong@tio.org.cn :,,, xuchangan@tio.org.cn : , :

11693/hyhz20150300072,,,,, (Vanittanakom et al, 1986; Burgess et al, 1993; Barsby et al, 2001; Vaseeharan et al, 2003;, 2011;, 2011;, 2012;, 2012;, 2012);,,,, LHB02 (Vibrio harveyi)

2 5 : FA ,,,,,,, LB : 10g, 5g, 10g, 1000 ml, ph SLB : 10g, 5g, 10g, 20g, 1000 ml, ph : 9.0 g 10.0 g, 5.0 g, 1000 ml CMC-Na : 15.0 g 1.0 g 1.0 g 1.0 g 1.0 g 20.0 g 1000 ml, ph 1.3 (Aeromonas hydrophila), (Edwardsiella), (Vibrio harveyi), (Vibrio alginolyticus), (Vibrio parahaemolyticus), (Staphylococcus aureus) 1.4, PCR,,, g, 100mL 0.85% NaCl, LB, 37 C, 180 r/min, 30h, 75 C 20min 1.5.2, : FA01 FA02 FA03 FA04 FA05 FA06 FA07 FA08 FA09 FA10, LB, 37 C, 180 r/min, 6 h, 10%, 37 C, 180 r/min, 48h, r/min, 6min,,, ; CMC-Na,, 50μL, 37 C ; 1h, 0.85% NaCl,, 1.6 (, 2001), 16S rdna SLB, 37 C, 24 h, 1.6.2, D- L- D- D- ; ; V-P S rdna (, 2011), DNA DNA, 16S rdna, 27F: 5 -AGAGTTTGATCCTGGCTCAG R: 5 -GGTTACCTTGTTACGACTT-3 Taq DNA PCR, PCR : 94 C 4min; 94 C 45s, 55 C 45s, 72 C 1 min, 30 ; 72 C 10 min ; 4 C SanPreP DNA PCR PCR 16S rdna Blast,, MEGA, 1.7 LB, 37 C, 180 r/min, 6h, 10%, 37 C, 180 r/min, 24h, r/min, 6min,,, (, 2012;, 2013), 3, 5- (DNS), ph ph ph (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0), 37 C, 1h,, 100%

85% NaCl, LB, 37 C, 180 r/min, 30h, 75 C 20min 1.5.2, : FA01 FA02 FA03 FA04 FA05 FA06 FA07 FA08 FA09 FA10, LB, 37 C, 180 r/min, 6 h, 10%, 37 C, 180 r/min, 48h, 10000 r/min, 6min,,, ; CMC-Na,, 50μL, 37 C ; 1h, 0.

3 C 1h,, 100% nm, 10cm min,, 100% , 5mL LB, 37 C, 180 r/min 16h, 1 ml 100 ml C SLB,, FA08 5mL LB, 37 C, 180 r/min 6 h, 10% 50 ml LB, 37 C, 180 r/min 17 h, r/min 6 min, (agar diffusion method) (Tagg et al, 1971) ( ) 2 2.1, FA08,, 1,, FA08 1 FA08 Fig.1 The hydrolysis circle of strain FA FA08 FA08 SLB, 37 C 24h,,,,,, 2.3 FA08 1 表 1 功能菌 FA08 的生理生化检测结果 Tab.1 Physiological and biochemical characteristics of strain FA08 D- + L- + D- + D V-P , S rdna 16S rdna, FA08 16S rdna 1407bp 2 (http: // gov), Blast,, ( 2), MEGA,, 3,, FA08 Bacillus licheniformis (X68416) Bacillus licheniformis (NR118959), Bacillus licheniformis (X68416) 99%, FA08 (Bacillus licheniformis), KM ph FA08 4, FA08 ph 4, ph 3 ph 5 10, 85%, FA08

3 FA08 1 表 1 功能菌 FA08 的生理生化检测结果 Tab.1 Physiological and biochemical characteristics of strain FA08 D- + L- + D- + D- + + - + + - V-P + - + +, - 2.

4 5 : FA , FA C, C 75%, 80 C 70%, FA08 6 FA08, 100%, 2 FA08 16S rdna Fig.2 The 16S rdna sequence of strain FA08 Fig S rdna Phylogenetic dendrogram based on 16S rdna sequence

5 表 2 选自基因库中用于构建系统发育树的菌株及其录入号 Tab.2 The strains and the accession numbers in GenBank for phylogenetic dendrogram construction X68416 NR X60633 NR NR NR X82436 NR NR NR NR NR NR FJ Bacillus licheniformis Bacillus licheniformis Bacillus popilliae Bacillus coahuilensis Virgibacillus chiguensis Virgibacillus kekensis Gracilibacillus dipsosauri Bacillus aryabhattai Bacillus xiaoxiensis Paenibacillus macquariensis Paenibacillus lentimorbus Pelosinus propionicus Pelosinus fermentans Bacillus amyloliquefaciens 2.6 FA08 : 3, FA08 :, 表 3 功能菌 FA08 发酵上清对病原菌的拮抗性能 Tab.3 Antimicrobial activity of FA08 s supernatant on selected pathogens (Aeromonas hydrophila) (Edwardsiella) (Vibrio harveyi) (Vibrio alginolyticus) (Vibrio parahaemolyticus) (Staphylococcus aureus) (mm) ph FA08 Fig.4 The effect of ph on the stability of cellulase produced by strain FA08 5 FA08 Fig.5 The effect of temperature on the stability of cellulase produced by strain FA08 6 FA08 Fig.6 The effect of UV on the stability of cellulase produced by strain FA08 (, 2008) 1989, FDA, ; (, 2011),,,,, (, 2014);,,, (, 2006);, (, 2005),,,, (, 2009) FA08,, FA08, FA08, ph3 ph %, (2012) (Bacillus),,, FA08

109390 FJ655812 Bacillus licheniformis Bacillus licheniformis Bacillus popilliae Bacillus coahuilensis Virgibacillus chiguensis Virgibacillus kekensis Gracilibacillus dipsosauri Bacillus aryabhattai

6 5 : FA , ( ) (, 2011), FA08,,,,, (, 2012),,, FA08, 80 C 70%, (2009),,,,,,,,, C, FA08,, : Wl0, (, 2013); c-4, (, 2009) FA08, FA08 :, FA08,,,,,, FA08, FA08,,,, FA08,,, , 41(17): ,,, , (2): 10 13,, 琤琤, , (12): 62 64,, :, 43 65,,, , 28(4): 80 84,,, , 21(4): ,,, (Carassius auratus gibelio)., 38(1): 29 35,, C-4., 36(5): ,,, Wl0., 29(4): ,,, , 39(4): ,,, , 40(14): ,,, , (17): 9 11,,, , 35(3): ,,, , 31(10): ,,, , 26(7): ,,, , (4): 73 74,,, , 41(5): 45 51,,, LHB02., 42(2): ,, , 51(7): ,,, , 22(10): ,,, 2008.

, 38(1): 29 35,, 2009. C-4., 36(5): 392 396,,, 2013. Wl0., 29(4): 579 585,,, 2008. 4., 39(4): 374 380,,, 2012.., 40(14): 8103 8105,,, 2012.., (17): 9 11,,, 2011.

7 , 47(3): ,, , (1): 17 19,,, , 19(1): 36 40,,, , (7) 14 18,,, , 18(1): 75 79,,, , 33(4): Barsby T, Kelly M T, Gagne S M et al, Bogorol a produced in culture by a marine Bacillus sp. reveals a novel template for cationic peptide antibiotics. Org Lett, 3(3): Burgess J G, Miyashita H, Sudo H et al, Gene transfer in Chromatium purpuratum, a marine photosynthetic bacterium producing antibiotics. J Mar Biotechnol, 1(2): 101 Liu K-F, Chiu C-H, Shiu Y-L et al, Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae. Fish Shellfish Immunol, 28(5 6): Tagg J R, McGiven A R, Assay system for bacteriocins. Appl Microbiol, 21(5): 943 Tamehiro N, Okamoto-Hosoya Y, Okamoto S et al, Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrob Agents Chemother, 46(2): Vanittanakom N, Loeffler W, Koch U et al, Fengycin-A novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F J Antibiotics, 39(7): Vaseeharan B, Ramasamy P, Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett App Microbiol, 36(2): SCREENING AND IDENTIFICATION, ENZYME CHARACTERISTICS, AND ANTIBACTERIAL ACTIVITY OF A MARINE BACILLUS STRAIN FA08 FANG Wei-Dong 1, 2, TANG Xu 3, LIU Yuan-Sen 3, LIN Ling 3, HUANG Shi-Xin 3, GAO Pei-Li 3, XU Chang-An 3 (1. Fisheries College, Jimei University, Xiamen , China; 2. Fujian Provincial Haixin Group Co., Ltd., Zhangzhou , China; 3. Third Institute of Oceanography, State Oceanic Administration, Xiamen , China) Abstract To develop biocontrol technology with bacterium against pathogens for prevention from aquatic diseases, we screened a bacillus strain FA08 from mud in an aquaculture area in Ningde, Fujian, China. The strain could produce with cellulase and showed antimicrobial activity, and has been identified as Bacillus licheniformis (GenBank Accession number KM892857), based on its 16S rdna sequence homology, together with the morphological, physiological and biochemical characteristics. Analysis of its enzyme characteristic showed that the cellulase could stand in a wide ph range from 3 to 10 and remain thermally stable in C with relative cellulase activity up to 70%. Moreover, the cellulase was insensitive to UV. In addition, we tested the antimicrobial activity of FA08 against selected aquatic pathogens. The results revealed that supernatant of the strain could inhibit strongly against Aeromonas hydrophila, Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus, and Staphylococcus aureus. Therefore, strain FA08 and its cellulase product shall have promising values of development and application for aquaculture. Key words Bacillus licheniformis; cellulase; screening and identification; enzymatic character; antimicrobial activity

Gene transfer in Chromatium purpuratum, a marine photosynthetic bacterium producing antibiotics. J Mar Biotechnol, 1(2): 101 Liu K-F, Chiu C-H, Shiu Y-L et al, 2010.

Microsoft Word - 16-夏小安-lb.doc

Microsoft Word - 16-夏小安-lb.doc 热带海洋学报 JOURNAL OF TROPICAL OCEANOGRAPHY 2010 年第 29 卷第 5 期 :103 110 海洋生物学 http://jto.scsio.ac.cn; http://www.jto.ac.cn 夏小安 1, 吴清洋 1, 李远友 1, 王树启 1, 游翠红 1, 林尤顺 (1., 515063; 2., 515000)