南海北部表层海水微生物多样性及部分真菌活性物质研究

Microbial diversity and active substances of some fungi in surface seawater of the northern South China Sea

为探究南海北部表层海水微生物的多样性及部分真菌代谢产物的活性,采取平板涂布稀释的方法对南海北部8个站位表层海水样品进行分离,基于16S rRNA和ITS序列测定对纯培养的微生物进行初步鉴定,共分离到162株细菌和43株真菌。其中162株细菌分属于4个门,5个纲,15个目,21个科,35个属的61个种,变形菌门和放线菌门为优势门;43株真菌分属于子囊菌门和担子菌门的5个纲,7个目,7个科,7个属的14个种,曲霉属为优势属。从分离得到的真菌中选取16株真菌进行培养,对其粗提物进行组蛋白去乙酰化酶抑制活性、抗肿瘤活性和抑菌活性筛选,结果表明:在100μg·mL^(-1)浓度下具有组蛋白去乙酰化酶抑制活性,抑制率在44%~54%之间;在60μg·mL^(-1)浓度下对人宫颈癌细胞的抑制率在2%~37%之间;在200μg·mL^(-1)浓度下对嗜水气单胞菌(Aeromonas hydrophila)和维氏气单胞菌(Aeromonas Vickers)两株指示菌未显示抑菌效果。研究结果表明,南海局部地区表层海水可培养微生物多样性高;对部分真菌的活性测试结果为后续筛选天然产物来源的海洋药物提供了一定的参考。

To survive in the complex ocean environment,marine microbes have evolved a variety of adaptation systems,including the production of specific biomolecules and unique bioactive compounds which were never found in terrestrial environments.Marine microorganisms with abundant species and unique metabolism are important sources of marine natural products and new medicines.The South China Sea(SCS)is the largest sea in China.The abundant microbial resources in SCS are ideal objects for studying marine microbial diversity and natural products.To study the microbial diversity of surface seawater in SCS and bioactivity of fungi extracts,a study was carried out.Surface seawater samples were collected from eight stations in the northern part of SCS,then were filtered and diluted to different concentrations and spread on bacterial and fungal plates,finally,the plates were cultured in a constant temperature incubator at 28℃ for 3-28 d.After cultivation,the morphological characteristics of the colonies were visually observed.Colonies of different forms were brought up in corresponding culture media after preliminary screening and purified into single colonies,then stored under 4℃ on an inclined plane.With a molecular protocol by DNA amplification and sequencing of the 16 S rRNA and ITS gene sequence alignment,a total of 162 bacterial strains and 43 fungal strains were isolated.The 162 bacterial strains belonged to 4 phyla,5 classes,15 orders,21 families,35 genera and 61 species.Proteobacteria and Actinomycetes were the dominant phylum.A total of 105 strains of Proteobacteria were identified as 84 strains of α-Proteobacteria,accounting for 51.8%;and 21 strains of γ-Proteobacteria,accounting for 12.9%;50 strains of Actinomycetes,accounting for 30.8%,4 strains of Bacteroidetes,accounting for 2.5%,3 strains of Firmicutes,accounting for 1.9%.Microbacterium and Erythrobacter were the dominant genera,which isolated 40 and 23 strains,respectively;E.pelagi was the highest with 12 strains.E.flavus,M.aurantiacum and M.schleiferi followed.From the results,the diversity and quantity of culturable bacteria isolated from different stations were different.Proteobacteria and actinobacteria were isolated from all 8 stations,and the number of proteobacteria strains was more than actinobacteria strains in each station,while Firmicutes and Bacteroidetes were only isolated from 3 stations.The bacteria genera diversity of stations 15,19 and 21 was the highest,while the station 17 was the least with only 4 genera.Erythrobacter,Microbacterium and Qipengyuania were distributed widely and isolated from seven stations.Twenty genera of bacteria,which accounting for 57% of the total genus numbers,were only isolated from specific stations.The bacterial strains isolated from Firmicutes were least,and all strains were classified to Bacillus.43 strains of fungi were isolated from eight stations,and ITS sequence results showed great similarity to fungi(over 98%) after comparison with database.The 43 strains were classified to 2 phyla,5 classes,7 orders,7 families,7 genera and 14 species.Strains isolated from Ascomycota were the highest(40 strains).The other three strains belonged to Basidiomycota.Aspergillus was the dominant genus among the strains,a total of 21 Aspergillus strains were isolated,accounting for 48.8% of 43 strains.In terms of number of species,Penicillium oxalicum was the most common species in which 9 strains were isolated,while Aspergillus versicolor followed with 7 strains.According to the fungal sequence results and the station difference of fungi,16 strains of fungi were selected for further small-scale fermentation.After incubation for 18 d,the cultures were ultrasonic-extracted two times with ethanol,respectively.After filtered and vacuum evaporated,the crude extracts were dissolved in DMSO to prepare 10 mg·mL^(-1)solutions for further activity screening.The HDAC inhibitor screening kit was used to screen the histone deacetylase inhibitory activity.Black 96-well plate was used and the total reaction system was 100 μL.SAHA was dissolved in DMSO to prepare a 100 μmol·L^(-1)SAHA solution as positive control 1;TSA was used as positive control 2.Three parallel replicates were set for each sample to calculate the inhibitory activity of HDAC.Compared with the positive control groups,the crude extracts of these 16 fungi showed a certain inhibitory effect on HDAC.A.sydowii b-96 showed the highest inhibition rate as 54%,while A.flavus f-111 showed the lowest as 44%.The inhibition rate to histone deacetylase of 8 fungi crude extracts,A.Sydowii B-96,A.versicolor H-291,A.versicolor E-118,and Cladosporium halotolerans B-61,Cystobasidium Minutum E-114,P.Oxalicum F-112,Cystobasidium Minutum E-129 and P.Oxalicum B-49,was measured between 50% and 54%.The other fungi crude extracts to HDAC ranged from 44% to 49%.The activity of crude extracts from 16 strains of fungi on HeLa cells was determined by MTT method.The fungal crude extract was diluted to 60 μg·mL^(-1),and the paclitaxel was set as positive control with concentration of 2 μg·mL^(-1).Three parallel replicates were set.Results showed that these 16 fungal crude extracts had weak activity from 2% to 37% at a concentration of 60 μg·mL^(-1).The crude extracts of P.oxalicum H-287 and A.versicolor H-291 showed the highest inhibition activity of Hela cells by 37% and 35%,respectively,which was similar to the results of previous literature.Secondary metabolites with anti-tumor activity from these two fungi were supposed to acquire after expansion of fermentation in future study.The antibacterial activity of crude extracts was determined by K-B disk method.The crude fungal extract was diluted to 0.2 mg·mL^(-1).Aeromonas Hydrophila and A.Vickers were inoculated into liquid medium for activation,then the bacterial solution concentration was adjusted to 104 cfu·mL^(-1)and coated on LB plate.The sterilized filter paper was placed on the plate,and 5 μL of diluted fungal crude extract was added.Then the plates were cultured in a constant temperature incubator at 28℃ for 12 h.The blank plate was used as negative control.Three parallel replicates were set for each sample to calculate diameter of antibacterial circle.Results showed that the 16 crude extracts did not show antibacterial activity against A.hydrophila and A.Vickers at the concentration of 200 μg·mL^(-1).According to literatures,secondary metabolites of some Aspergillus fungi had bacteriostatic effect on aquatic pathogens such as A.hydrophila.In our research,no crude extracts of these 16 fungi showed bacteriostatic effect on the same bacteria.And because the test concentration in this experiment is set as a single concentration,it is speculated that the concentration of active secondary metabolites in crude extracts is lower than its minimum inhibitory concentration.More groups of different extracts concentration should be set in further study to testify the bacteriostatic activity.Results showed that the microbial diversity of surface seawater in SCS was high and the extracts had multi-bioactivity.And it will provide references for developing and using of fungi sources of SCS in the future.