: 379 (Bacillus thuringiensis) (Bacillus subtilis) (Salmonella typhi) (Shigella flexneri) (Proteus vulgaris) (Pseudomonas aeruginosa) (Enterobacter ae

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1 微生物学通报 MAR 20, 2008, 35(3): 378~383 Microbiology 研究报告 2008 by Institute of Microbiology, CAS 一株具有广谱抗菌活性小单孢菌的分离和鉴定 * 龙中儿朱跃进黄运红付学琴 ( ) 摘要 : 从南昌瑶湖的农田土壤样品中分离到一株具有广谱抗菌活性的稀有放线菌通过形态特征培养特征生理生化特性细胞化学成分及 16S rrna 基因序列等多相分类特征研究, 将该稀有放线菌鉴定为炭样小单孢菌关键词 : 稀有放线菌, 分类鉴定, 16S rrna 基因, 炭样小单孢菌 Isolation and Identification of a Strain of Micromonospora with Broad-spectrum Antimicrobial Activity LONG Zhong-Er * ZHU Yue-Jin HUANG Yun-Hong FU Xue-Qin (College of Life Science, Jiangxi Normal University, Nanchang ) Abstract: A rare strain of actinomycetes, with broad-spectrum antimicrobial activity, was isolated from the soil samples from the farmland in the area of Yaohu lake in Nanchang. The information about the taxonomic identification, such as the morphology, physiological properties, cell components and 16S rrna gene sequences, suggested that the rare strain of actinomycetes was identified as Micromonospora carbonacea. Keywords: Rare actinomycetes, Taxonomic identification, 16S rrna gene, Micromonospora carbonacea [1],, [2 3], ( 5% ), [4], [5 8], 1 材料与方法 1.1 材料土壤样品 : 1, 10 cm,, 200 g,,, 活性检测靶菌 : (Staphylococcus aureus) (Sarcina lutea) 基金项目 : (No. [2007]133 ) * 通讯作者 :Tel: ; : [email protected] 收稿日期 : ; 接受日期 :

2 : 379 (Bacillus thuringiensis) (Bacillus subtilis) (Salmonella typhi) (Shigella flexneri) (Proteus vulgaris) (Pseudomonas aeruginosa) (Enterobacter aerogenes) (Escherichia coli) (Saccharomyces cerevisiae) (Penicillium chrysogenum) (Rhizopus oryzae) [9] 制霉菌素高氏 1 号培养基 : 1 (50 /, ) 45 IU/mL-55 IU/mL 1 ml 1000 ml 1, 1 [10,11] 营养琼脂培养基 :, 发酵培养基 : 45 g, 25 g, K 2 HPO g, NaCl 1 g, Na 2 SO g, FeSO g, CaCO 3 3 g, 1000 ml, ph7.2, Pa 20 min 活性检测培养基 : 20 g, 1000 ml, Pa 20 min ; 5 g /L, 1.2 方法稀有放线菌的分离 :, h [11], 1%(W/V), 0.2 ml 1, 28 7 d,,, 产抗生素稀有放线菌的筛选 : 1 50 ml 250 ml, r/min 4 d, 4000 r/min 20 min,, 4 [9],,, 形态观察 : 1 (28 ), 21 d( Motic / DM-BA200, 130 Motic, Motic Digital Class 1.1 Motic Images Advanced 3.1 ) ( S-570 ) 生长温度检测 : 1, d 14 d, 培养特征和生理生化特性 : [12] 细胞化学组分分析 : [12,13] S rrna 基因的 PCR 扩增及其序列分析 : [14] DNA, PCR 16S rrna PCR [15] Pf 5 -AGAGTTTGATCATGGCTCAG-3 ; Pr 5 -TACGGCTACCTTGTTACGACT-3 PCR 94 5 min; 94 1 min, 57 1 min, 72 2 min, 35 ; min PCR 基于 16S rrna 基因序列的系统发育分析 : 16S rrna, Blast GenBank 16S rrna, Clustal X [16], PHYLIP [17,18], TreeView [18], DNAman 2 结果与分析 2.1 稀有放线菌的分离及其抗菌活性, 1, JXNU-1, 1,

3 380 微生物学通报 2008, Vol.35, No.3 表 1 稀有放线菌 JXNU-1 发酵液的抗菌活性 Table 1 Antimicrobial activity of fermented broth from the rare strain of actinomycetes JXNU-1 Strain for activity detection (mm) Diameter of inhibiting zone (mm) (Staphylococcus aureus) 20.8 (Sarcina lutea) 20.2 (Bacillus thuringiensis) 18.0 (Bacillus subtilis) 19.8 (Salmonella typhi) 21.2 (Shigella flexneri) 20.8 (Pseudomonas aeruginosa) 20.2 (Saccharomyces cerevisiae) 20.8 (Proteus vulgaris) 0 (Enterobacter aerogenes) 0 (Escherichia coli) 0 (Penicillium chrysogenum) 0 (Rhizopus oryzae) 0,,, 2.2 形态特征 JXNU-1 1,, 4 d, 5 d, 21 d,,,,, ; ;,, ( 7 d 1, 2 ) (a) (b) Fig. 1 图 1 稀有放线菌 JXNU-1 的基内菌丝 (a) 和孢子 (b)( 1000 倍 ) Morphological characteristics of the rare strain of actinomycetes JXNU-1 grown on Gause 1 medium for 7d at 28, showing the substrate mycelium (a) and spora (b) ( 1000) 图 2 稀有放线菌 JXNU-1 孢子的扫描电镜图 ( 8000) Fig. 2 Scanning electron micrograph of the spores of the rare strain of actinomycetes JXNU-1 grown on Gause 1 medium for 7d at 28 ( 8000) 2.3 培养特征 JXNU-1 1, 28 37, 45 (28 ) 2,,, 2.4 生理生化特征 JXNU-1,,,, ; - L- D- β- D-

4 : 381 Table 2 表 2 稀有放线菌 JXNU-1 的培养特征 Cultural characteristics of the rare strain of actinomycete JXNU-1 (Medium) (Colour of mycelium) (Growth state of mycelium) (Soluble pigment) (Colour of spore-bearing mycelium) - - D- D-, L- 2.5 细胞化学组分分析 JXNU-1,,, ; meso-dap, 2.6 基于 16S rrna 基因序列的系统发育分析 JXNU-1 16S rrna, 1479 ( GenBank database EF583904) GenBank database Micromonospora 16S rrna 1000, 分类鉴定结果分析, [19], JXNU-1 16S rrna, JXNU-1 Micromonospora carbonacea subsp. carbonacea Micromonospora carbonacea subsp. aurantiaca, 99% ;,,, JXNU-1 Micromonospora carbonacea subsp. carbonacea Micromonospora carbonacea subsp. aurantiaca ( 3 ), JXNU-1 Fig. 3 图 3 以 16S rrna 基因序列同源性为基础的稀有放线菌 JXNU-1 系统发育树 Phylogenetic tree of the rare strain of actinomycete JXNU-1 based on the sequence of 16S rrna gene

5 382 微生物学通报 2008, Vol.35, No.3 表 3 稀有放线菌 JXNU-1 与亲缘关系相近的两个菌株的生理生化特征的比较 Table 3 Comparison of physiological characteristics between the rare strain of actinomycetes JXNU-1 and sibship-similar strains (Physiological characteristics) JXNU-1 Micromonospora carbonacea subsp Carbonacea Micromonospora carbonacea subsp aurantiaca (Gelatin liquefaction) (Amylohydrolysis) (Milk solidation) (Milk peptonization) (Cellulose hydrolysis) (Nitrate reduction) (Melanin generation) H 2 S (H 2 S generation) ND ND - ( -melibiose) (Raffinose) L- (L-rhamnose) L- (L-arabinose) (Glucose) D- (D-galactose) - ( -lactose) D- (D-fructose) (Sucrose) D- (D-xylose) (Glycerol) D- (D-ribose) : ; : ; ND: : Positive; : Negative; ND: Not determined 3 结论 , 45, ;,, ;, ;, ;,,,, ; Ⅱ ; 16S rrna Micromonospora carbonacea subsp. carbonacea Micromonospora carbonacea subsp. aurantiaca ;, 参考文献 [1].., 2002, 27(2): [2] Fiedler HP, Bruntner C, Bull AT, et al. Marine actinomycetes as a source of novel secondary metabolities. Antonie van Leeuwenhock, 2005, 87(1): [3],.., 2005, 21(6): [4],.., 2002, pp [5] Peric-Concha N, Long PF. Mining the microbial metabolome: a new frontier for natural product lead discovery. Drug Discovery Today, 2003, 8(23): [6] Donadio S, Monciardini P, Alduina R, et al. Microbial technologies for the discovery of novel bioactive metabolities. Journal of Biotechnology, 2002, 99(3): [7] Lazzarini A, Cavaletti L, Toppo G, et al. Rare genera of actinomycetes as potential producers of a new antibiotics. Antonie van Leeuwenhock, 2000, 78(3-4):

6 : 383 [8] Shomura T. Screening for new products of new species of Dactylosporangium and other actinomycetes. Actinomycetology, 1993, 7(2): 88. [9]..., 1999, pp , [10],,,.., 2000, 17(2): [11],,.., 2002, 23(1): [12], 1990, pp [13].., 1986, 13(5): [14]. Streptomycetes [15] Minciardini P, Sosio M, Cavaletti L, et al. New PCR primers for the selective amplification of 16S rdna from different groups of actinomycetes. Microbiology Ecology, 2002, 42: [16] Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL-X windows interface: flexable strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids research, 1997, 25(24): [17] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol, 1987, 4: [18] Stackebrandt E, Rainey FA, Ward-rainey NL. Proposal for new hierarchic classification system, actinobacteria classis nov. International journal of systematic bacteriology, 1997, 47(2): [19] RE, NE... :, 1984, pp 中国科学院微生物研究所期刊广告部成立编辑部公告 , 8107,,,, CA BA MEDLINE AJ, PCR,,,, , 提示 : 2007, 收款单位 : 开户银行 : 帐号 : 联系电话 : 联系人 : 电子信箱 :[email protected] 网址 :

218 微生物学通报 2009, Vol.36, No 活性检测菌 : Staphylococcus aureus CMCC Escherichia Coli CMCC Pseudomonas aeruginosa CMCC Bacillus su

218 微生物学通报 2009, Vol.36, No 活性检测菌 : Staphylococcus aureus CMCC Escherichia Coli CMCC Pseudomonas aeruginosa CMCC Bacillus su 微生物学通报 FEB 20, 2009, 36(2): 217~222 Microbiology [email protected] 2009 by Institute of Microbiology, CAS 研究报告一株杀黄孢链霉菌 IMB-14 的分离与鉴定 * 邹懿刘莉胡昌华 ( 400716) 摘要 : 采用薄膜过滤法从武当山采集土壤中分离到一株具有广谱抗菌活性的链霉菌, 命名为 IMB-14