智利竹鱼渔场海洋微生物的物种多样性及其生物活性筛选
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摘要
利用东海水产研究所开展的2012年东南太平洋智利竹鱼(Trachurus murphyi)渔场生态环境调查,对该海域75个站点进行了海洋微生物样品的采集;通过菌株的16S rRNA测序及GenBank数据库比对,分析了从所采样品中分离获得的海洋微生物菌株的物种多样性;通过菌株发酵、代谢产物提取及其抗菌滤纸扩散分析,对其发酵代谢产物粗提物进行了抗菌活性筛选;通过类胡萝卜素合成通路中八氢番茄红素合成酶(crtB)及脱氢酶(crtI)基因分析,对筛选到的抗菌活性菌株进行了基因筛选。结果表明,累计分离获得可培养海洋微生物菌株628株,其隶属12个属,其中的优势菌属包括亚硫酸盐杆菌属(Sulfitobacter)、弧菌属(Vibrios)及气单胞菌属(Aeromonas)。其中16株菌株的发酵代谢产物具有明显的抗菌活性,而其中12株活性菌株具有合成类胡萝卜素的crtB、crtI分子遗传基础。
Exploration of new marine microbial resources is a highlight in fields like development and utilization of marine biological resources,and innovative marine drugs. And research on mining,storage and function of new marine microbial resources in pelagic fishery will open a new way for substantial development and utilization of marine resources in specific areas. Trachurus murphyi Chile fishery in the southeast Pacific is an important part of pelagic fishery for China. This study is to isolate and purify marine microbial strains from samples of Trachurus murphyi Chile fishing grounds,analyze their microbial biodiversity and screen their bioactivity. Environmental microbial samples from 75 sites were obtained through ecological and environmental investigation on Trachurus murphyi Chile fishing grounds in 2012. The microbial biodiversity analysis of isolated stains was performed using 16 S rRNA gene sequencing and gene homology blasting performed in GenBank. The following bioactive strains screening for anti-bacterial bioactivity was performed using microbial strain fermentation, fermentation extraction and paper filter diffusion analysis using Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Vibro parahaemolyticus as indicators. The molecular gene analysis was accomplished focusing on two key genes,phytoene synthase( crtB) and phytoene desaturase( crtI),during the carotenoid biosynthesis pathway. In total 628 microbial strains were isolated,and 12 genuses were indentified,and the predominant bacteria were Sulfitobacter,Vibrios and Aeromonas. All 20 screened active strains could produce pigments and colors of their bacterial colony,including black,pink,red and yellow.Their cell diameters ranged from 0. 9 to 1. 2 μm,and could utilize various carbon sources to grow. Results of16 S rRNA gene sequencing and gene homology blasting performed in GenBank for No. 522 strain showed that it had 97. 1% gene similarity with the previously reported strains,indicating that it was a potentially new species,and was nominated as Ahrensia sp. ZJY-522. Moreover,No. 529 and No. 601 strains were found to belong to the genus of Alcanivorax,which were the dominant oil degrading microbes in the ocean. The bacteria in the genus of Alcanivorax had specific hydrocarbon degrading ability that could utilize the alkanes with different long chains,branches or straight chains. Results of bioactive strains screening,totally 16 strains demonstrated obvious antibacterial bioactivity,accounting for 2. 5% of total isolated strains. Among them,Alcanivorax sp. ZJY-522 demonstrated the most obvious anti-bacterial bioactivity, especially for two indicators,Escherichia coli and Staphylococcus aureus. Currently,the scale fermentation,extraction of active compounds produced by this strain and their chemical structure analysis are in progress. Based on the results by gene screening,12 strains were found to have genetic basis of crtB and crtI for the carotenoid biosynthesis.The study will lay a basis for further research on development and utilization of marine resources in Trachurus murphyi Chile fishing grounds,and for the discovery of new lead compounds of marine drugs in the future.
Exploration of new marine microbial resources is a highlight in fields like development and utilization of marine biological resources,and innovative marine drugs. And research on mining,storage and function of new marine microbial resources in pelagic fishery will open a new way for substantial development and utilization of marine resources in specific areas. Trachurus murphyi Chile fishery in the southeast Pacific is an important part of pelagic fishery for China. This study is to isolate and purify marine microbial strains from samples of Trachurus murphyi Chile fishing grounds,analyze their microbial biodiversity and screen their bioactivity. Environmental microbial samples from 75 sites were obtained through ecological and environmental investigation on Trachurus murphyi Chile fishing grounds in 2012. The microbial biodiversity analysis of isolated stains was performed using 16 S rRNA gene sequencing and gene homology blasting performed in GenBank. The following bioactive strains screening for anti-bacterial bioactivity was performed using microbial strain fermentation, fermentation extraction and paper filter diffusion analysis using Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Vibro parahaemolyticus as indicators. The molecular gene analysis was accomplished focusing on two key genes,phytoene synthase( crtB) and phytoene desaturase( crtI),during the carotenoid biosynthesis pathway. In total 628 microbial strains were isolated,and 12 genuses were indentified,and the predominant bacteria were Sulfitobacter,Vibrios and Aeromonas. All 20 screened active strains could produce pigments and colors of their bacterial colony,including black,pink,red and yellow.Their cell diameters ranged from 0. 9 to 1. 2 μm,and could utilize various carbon sources to grow. Results of16 S rRNA gene sequencing and gene homology blasting performed in GenBank for No. 522 strain showed that it had 97. 1% gene similarity with the previously reported strains,indicating that it was a potentially new species,and was nominated as Ahrensia sp. ZJY-522. Moreover,No. 529 and No. 601 strains were found to belong to the genus of Alcanivorax,which were the dominant oil degrading microbes in the ocean. The bacteria in the genus of Alcanivorax had specific hydrocarbon degrading ability that could utilize the alkanes with different long chains,branches or straight chains. Results of bioactive strains screening,totally 16 strains demonstrated obvious antibacterial bioactivity,accounting for 2. 5% of total isolated strains. Among them,Alcanivorax sp. ZJY-522 demonstrated the most obvious anti-bacterial bioactivity, especially for two indicators,Escherichia coli and Staphylococcus aureus. Currently,the scale fermentation,extraction of active compounds produced by this strain and their chemical structure analysis are in progress. Based on the results by gene screening,12 strains were found to have genetic basis of crtB and crtI for the carotenoid biosynthesis.The study will lay a basis for further research on development and utilization of marine resources in Trachurus murphyi Chile fishing grounds,and for the discovery of new lead compounds of marine drugs in the future.
引文
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[2]SUTTON T T.Vertical ecology of the pelagic ocean:classical patterns and new perspectives[J].J.Fish Biol.,2013,83(6):1508-1527.
[3]STOCKER R.Marine microbes see a sea of gradients[J].Science,2012,338(6107):628-633.
[4]BLUNT W J,COPP B R,MUNRO M H G,et al.Marine natural products[J].Nat.Prod.Rep.,2010,27(2):165-237.
[5]SCHMIDT E W,DONIA M S.Life in cellulose houses:symbiotic bacterial biosynthesis of ascidian drugs and drug leads[J].Curr.Opin.Biotechnol.,2010,21(6):827-833.
[6]SIEG R D,KUBANEK J.Chemical ecology of marine angiosperms:opportunities at the interface of marine and terrestrial systems[J].J.Chem.Ecol.,2013,39(6):687-711.
[7]XIONG Z Q,WANG J F,HAO Y Y,et al.Recent advances in the discovery and development of marine microbial natural products[J].Mar.Drugs,2013,11(3):700-717.
[8]黄洪亮,陈雪忠.东南太平洋智利竹鱼中层拖网捕捞技术[J].中国水产科学,2005,12(1):99-103.
[9]张衡,樊伟.2009年秋冬季东南太平洋智利竹鱼的渔业生物学特征[J].海洋渔业,2010,16(3):340-344.
[10]OLIVA M E.Metazoan parasites of the jack mackerel Trachurus murphyi(Teleostei,Carangidae)in a latitudinal gradient from South America(Chile and Peru)[J].Parasite,1999,6(3):223-230.
[11]张晓玲,杨桥,吴文惠.产贝毒裸甲藻共生海洋细菌ECSYW-28的分离鉴定及产类胡萝卜素生物活性研究[J].食品科学,2010,31(23):198-203.
[12]GONZALEZ L,SANDOVAL H,SACRISTAN N,et al.Identification of lactic acid bacteria isolated from Genestoso cheese throughout ripening and study of their antimicrobial activity[J].Food Control,2007,18(6):716-722.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:349-399.
[14]BUCHANAN R E,GIBBONS N E.伯杰细菌鉴定手册[M].第8版.北京:科学出版社,1984:729-759.
[15]GARRITY G M,BELL J A,LILBURN T G.Taxonomic outline of the prokaryotes.Bergey’s Manual of Systematic Bacteriology[M].2nd ed.,New York:Spring-Verlag,2004:4-10;210-212.
[16]张晓玲,杨桥,樊成奇,等.一株南极大磷虾共附生细菌的分离鉴定及一氧化氮靶向抑制活性筛选[J].海洋渔业,2011,33(4):436-441.
[17]LI A,SHAO Z.Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5[J].PLoS One,2014,9(2):e89144.
[18]PHAM N T A,ANONYE B O.Vying over spilt oil[J].Nat.Rev.Microbiol.,2014,12(3):156.
[19]ZHANG L,YANG Q,LUO X S,et al.Knock out of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability[J].Arch.Microbiol.,2007,188(4):411-419.