中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性

Diversity of culturable extracellular proteases producing marine fungi isolated from the intertidal zone of Naozhou Island in South China Sea

摘要:

【目的】研究中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性。【方法】从中国南海硇洲岛潮间带采集海水和沉积物样品，采用分离培养、蛋白酶生产菌平板检测法和基于内转录间隔区1-5.8S rRNA基因内转录间隔区2 (ITS1-5.8S-ITS2)序列的系统进化分析法，研究产胞外蛋白酶真菌的多样性。【结果】采用50%海水配制的马铃薯葡萄糖琼脂(PDA)固体培养板从所采集的样品中分离、纯化并收集了198株真菌分离株，并采用ITS1-5.8S-ITS2序列PCR扩增、测序、BLAST和系统进化分析的方法成功鉴定了其中的178株。其中，有10株的ITS1-5.8S-ITS2序列与其在NCBI数据库中最匹配的ITS1-5.8S-ITS2序列的一致性<97，表明它们有可能是新的物种；其余168株的ITS1-5.8S-ITS2序列与NCBI数据库中已存在的相关ITS1-5.8S-ITS2序列的一致性均≥98%。这178株真菌归属为66个种，分布在子囊菌门和担子菌门的6纲，16个目，27个科的45个属中。其中的主要属为青霉菌属，占28.70%；其次为曲霉属，占11.24%。有83株真菌在加在脱脂奶粉的PDA固体培养板上的菌落周围有一透明圈，表明其可产生分泌胞外蛋白酶。【结论】从中国南海硇洲岛潮间带共分离、鉴定和收集了178株真菌，其中10株可能是新的物种，83株为胞外蛋白酶生产菌。

Abstract:

[Objective] to investigate the diversity of marine fungi producing extracellular protease isolated from the intertidal zone of Naozhou Island in the South China Sea. [Methods] The samples of both seawater and sediment were collected from the intertidal zone of Naozhou Island in South China Sea, and used to investigate the diversity of the fungi producing extracellular proteases by culture-dependent method, protease producer plate assay, and a phylogenetic analysis based on the sequences containing internal transcribed spacer 1, 5.8S rRNA gene and internal transcribed spacer 2 (ITS1-5.8S-ITS2). [Results] A total of 198 isolates of fungi were isolated, purified and collected from the samples on the plates of Potato dextrose agar (PDA) medium prepared with 50% seawater. Amongst these isolates, 178 strains were identified successfully by PCR amplification, sequencing, BLAST and phylogenetic analysis of the ITS1-5.8S-ITS2 sequences. All these sequences showed ≥98% identity with the existing relative ITS1-5.8S-ITS2 sequences in the GenBank database, except 10 strains showed the identity <97% with their closest match suggesting their possibility of being novel species. These 178 strains represented 66 species, belonging to 45 genera of 27 families in 16 orders, 6 classes, spreading in two phyla, Ascomycota and Basidiomycota. The predominant genus was Penicillium, which takes a great proportion of 28.70%, followed by Aspergillus, which takes a proportion of 11.24%. There were 83 identified strains showed clear zone around their colonies on the PDA plates supplemented with skim milk indicating they could produce extracellular proteases. [Conclusion] A total of 178 strains of fungi were isolated and identified from the intertidal zone of Naozhou Island in the South China Sea. Amongst these strains, 10 strains would be novel specie Candidates, and 83 strains could produce extracellular proteases.