珊瑚共附生可培养真菌菌群多样性及其生物活性研究
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摘要
本文从广东徐闻珊瑚礁国家级自然保护区采集8种珊瑚,用培养法分离纯化真菌,应用形态学观察与分子鉴定相结合的方法,进行珊瑚共附生真菌菌群多样性研究,并对分离的真菌菌株进行生物活性筛选。
运用稀释平板法和基于internal transcribed spacer-rDNA (ITS-rDNA)基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究,将所得到的基因序列与NCBI数据库GenBank的序列进行相似性比较并构建系统发育树。实验中从8种珊瑚表面和内部共分离得到97株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明,珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中,4-13与GenBank中已报道的基因序列的相似性仅为89%,可能是潜在的新属种。
选取04丛生盔形珊瑚(Galaxea fascicularis)共附生可培养真菌进行抑菌活性和酶抑制活性筛选。采用96孔板培养法结合酶标仪测定。以6种常见的致病菌或致食品腐败的细菌为指示菌,对其共附生真菌次级代谢产物进行抗菌活性筛选,对抗菌能力及抗菌谱进行研究。结果表明:19株真菌均具有不同程度的抗菌活性。高活性菌株占52.63%,其中4-4、4-5两菌株表现出良好的抗菌活性。在酶抑制性实验中,有10株真菌对一种或两种酶具有抑制活性,其中曲霉属真菌效果良好,散囊属真菌4-12、枝孢霉4-20对乙酰胆碱酯酶的抑制效果超过了阳性对照石杉碱甲。粗提物样品对α-葡萄糖苷酶、乙酰胆碱酯酶有较好的抑制活性,说明具有抗老年痴呆、抗高血压活性。这对新药的研究开发提供了一定的理论基础,具有进一步的研究价值。结果表明,珊瑚共附生可培养真菌多样性较为丰富,潜在新的微生物菌种资源,并具有较好的生物活性,因此珊瑚共附生真菌具有良好的应用潜力和综合开发前景。
In this paper, we collect coral samples from Xuwen Coral National Nature Reserve area, identify fungus by morphological and molecular methods,in order to the preliminary investigation of species and bioactivity of coral symbiotic fungi.
To study the Phylogenetic diversity of coral cultured symbiotic fungi. By dilution plate technique and ITS-rDNA sequence analysis to research the diversity of the cultured symbiotic fungi with the coral, determined sequences data were submitted to GenBank and compared with those known sequences. Phylogenetic tree were built up with MEGA4.0 program. 97 different morphological strains were isolated from surface and inner of the 8 Stony corals. ITS-rDNA sequences analysis showed that, symbiosis cultured fungi mainly included Aspergillus sp., Cladosporium sp., Xylariales sp., Penicillium sp., Stachybotrys sp., Gibberella moniliformis, Fusarium sp. etc. The similarity of strain 4-13 sequence to the most closely related sequences in GenBank was only 89%, May be potential new species.
Select the cultured symbiotic fungi of 04 Galaxea fascicularis for antibacterial activity and enzyme inhibition activity screening. The microplate reader is used to detect the antibacterial activity of secondary metabolites of symbiosis fungi against pathogenic bacteria in sterile 96-well microtitre plates. The results showed that all symbiosis fungal strains have antibacterial activity where 52.63% fungal have higher antibacterial activity among which 4-4, 4-5 strains have broad-spectrum and strong antibacterial activity to indicator bacteria.In the enzymes inhibitory experiments, there are 10 strains fungi to one or two enzymes has inhibited activity. Aspergillus sp.has good effect. Enzyme inhibition activity of Eurotium rubrum4-12 and Cladosporium sp.4-20 are better than positive control. The good enzyme inhibition activity of Crude extractings provides certain theoretical for antidementia, antihypertensive new drugs studying, deserved for further study.
It is concluded that coral symbiotic fungi are potential new microorganism resources, have wide prospect of development.
运用稀释平板法和基于internal transcribed spacer-rDNA (ITS-rDNA)基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究,将所得到的基因序列与NCBI数据库GenBank的序列进行相似性比较并构建系统发育树。实验中从8种珊瑚表面和内部共分离得到97株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明,珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中,4-13与GenBank中已报道的基因序列的相似性仅为89%,可能是潜在的新属种。
选取04丛生盔形珊瑚(Galaxea fascicularis)共附生可培养真菌进行抑菌活性和酶抑制活性筛选。采用96孔板培养法结合酶标仪测定。以6种常见的致病菌或致食品腐败的细菌为指示菌,对其共附生真菌次级代谢产物进行抗菌活性筛选,对抗菌能力及抗菌谱进行研究。结果表明:19株真菌均具有不同程度的抗菌活性。高活性菌株占52.63%,其中4-4、4-5两菌株表现出良好的抗菌活性。在酶抑制性实验中,有10株真菌对一种或两种酶具有抑制活性,其中曲霉属真菌效果良好,散囊属真菌4-12、枝孢霉4-20对乙酰胆碱酯酶的抑制效果超过了阳性对照石杉碱甲。粗提物样品对α-葡萄糖苷酶、乙酰胆碱酯酶有较好的抑制活性,说明具有抗老年痴呆、抗高血压活性。这对新药的研究开发提供了一定的理论基础,具有进一步的研究价值。结果表明,珊瑚共附生可培养真菌多样性较为丰富,潜在新的微生物菌种资源,并具有较好的生物活性,因此珊瑚共附生真菌具有良好的应用潜力和综合开发前景。
In this paper, we collect coral samples from Xuwen Coral National Nature Reserve area, identify fungus by morphological and molecular methods,in order to the preliminary investigation of species and bioactivity of coral symbiotic fungi.
To study the Phylogenetic diversity of coral cultured symbiotic fungi. By dilution plate technique and ITS-rDNA sequence analysis to research the diversity of the cultured symbiotic fungi with the coral, determined sequences data were submitted to GenBank and compared with those known sequences. Phylogenetic tree were built up with MEGA4.0 program. 97 different morphological strains were isolated from surface and inner of the 8 Stony corals. ITS-rDNA sequences analysis showed that, symbiosis cultured fungi mainly included Aspergillus sp., Cladosporium sp., Xylariales sp., Penicillium sp., Stachybotrys sp., Gibberella moniliformis, Fusarium sp. etc. The similarity of strain 4-13 sequence to the most closely related sequences in GenBank was only 89%, May be potential new species.
Select the cultured symbiotic fungi of 04 Galaxea fascicularis for antibacterial activity and enzyme inhibition activity screening. The microplate reader is used to detect the antibacterial activity of secondary metabolites of symbiosis fungi against pathogenic bacteria in sterile 96-well microtitre plates. The results showed that all symbiosis fungal strains have antibacterial activity where 52.63% fungal have higher antibacterial activity among which 4-4, 4-5 strains have broad-spectrum and strong antibacterial activity to indicator bacteria.In the enzymes inhibitory experiments, there are 10 strains fungi to one or two enzymes has inhibited activity. Aspergillus sp.has good effect. Enzyme inhibition activity of Eurotium rubrum4-12 and Cladosporium sp.4-20 are better than positive control. The good enzyme inhibition activity of Crude extractings provides certain theoretical for antidementia, antihypertensive new drugs studying, deserved for further study.
It is concluded that coral symbiotic fungi are potential new microorganism resources, have wide prospect of development.
引文
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