中国南海海绵可培养共生真菌种群多样性、抑菌活性的研究及PKS,NRPS功能基因的筛选
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摘要
采用马丁氏培养基、MYPG培养基、查氏培养基、高氏一号培养基、2216E培养基和M1培养基共6种不同培养基,分别在20℃、25℃、30℃温度下对采集自中国南海的10种海绵共生真菌进行分离培养。并根据18S rDNA片段序列信息对分离到的真菌菌株进行了系统发育分析。采用了牛津杯法以大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus sp.)、金黄色葡萄球菌(Staphlococcus aureus)、荧光假单胞菌(Pseudomonas fluorescens)、黑曲霉(Aspergillus nigers)和白假丝酵母(Candida albicans)为指标菌,对分离到的菌株进行抑菌活性的筛选,并用PCR技术对分离到的177株真菌基因组进行了聚酮合酶(PKS)基因和非核糖体肽合成酶(NRPS)基因的筛选。
实验结果显示:采用6种培养基3个温度梯度的组合共分离得到177株真菌菌株,其中从X4海绵中分离到了8株真菌菌株,X5(23)、X6(32)、X7(1)、X8(4)、X9(35)、X10(22)、X11(17)、X12(25)、X13(10)。在20℃、25℃、30℃三个培养温度下分离到的真菌菌株数量没有明显差异,在所采用的6种培养基中,马丁氏培养基和MYPG培养基分离得到的菌株数最多,分别为46和44;其次是查氏和M1培养基,分别为32和24;高氏一号培养基和2216培养基再次之,分别为17和14。采用真菌18S rDNA特异引物PCR扩增,通过TaqI、HinfI、HaeIII三种限制性内切酶对PCR产物进行RFLP酶切分型,共得到24株独立菌。对24株菌的PCR产物测序并BLAST进行在线序列比对,从而得到这些菌株的生物学信息。24株菌的序列分析显示,所有分离出的真菌分别属于子囊菌门下Ascomycota 4目(小囊菌目Microascales、黑痣菌目Phyllachorales、肉座菌目Hypocreales和散囊菌目Eurotiales)10属。其中散囊菌目下的青霉属(Penicillium)和曲霉属(Aspergillus()37.5%);肉座菌目下的棒束孢属(Isaria)、拟青霉属(Simplicillium)等6个属(41.6%)是优势的真菌类群。
采用6种指标菌对分离得到的177株真菌菌株进行抑菌活性筛选,结果共有11株真菌表现出了抑菌活性,其中3株(x4-167、x11-75、x12-91)对枯草芽孢杆菌(Bacillus sp.)表现出抑菌活性,8株(x6-13、x10-49、x10-63、x12-94、x12-95、x12-109、x13-105、x13-118)对荧光假单胞菌(Pseudomonas fluorescens)表现出抑菌活性,其中x13-105, x12-94和x13-118同时对枯草芽孢杆菌(Bacillus sp.)和荧光假单胞菌(Pseudomonas fluorescens)表现出活性。获得了15条790 bp的PKS基因片段, BLAST比对结果表明这些PKS基因片段与一株产黄青霉Penicillium chrysogenum Wisconsin 54-1255的基因组序列和一株致病烟曲霉Aspergillus fumigatus Af293的PKS序列具有很高的相似性(98%-99%)。研究中筛选到的PKS基因均为I型的PKS。同时,克隆到4条1200 bp的NRPS基因片段, BLAST比对结果表明4条NRPS序列都与产黄青霉Penicilliumchrysogenum Wisconsin 54-1255的基因组序列具有很高的相似性(98%-99%)。
本研究通过分子鉴定和系统发育的分析揭示南海海绵具有非常丰富多样的可培养真菌,是丰富的海洋微生物资源。相对于其它培养基,马丁氏培养基与MYPG培养基更适合海绵真菌的分离培养。不同的海绵体内真菌组成存在差异,海绵也存在富含真菌的海绵与寡真菌海绵之分。本研究揭示了青霉和曲霉属真菌在海绵中的广泛分布。作为海洋真菌活性产物中产生最多的来源,本研究分离得到的部分青霉和曲霉菌表现出了对细菌的广谱抑菌活性,从一些菌株中发现了PKS和NRPS基因的存在,暗示我们这些带有PKS和NRPS的真菌可能在海绵化学联合防御中起作用。在对真菌PKS和NRPS研究中我们发现,在同一菌株中存在多种PKS或NRPS基因,基于PKS和NRPS基因的多样性和新颖性,可以指导筛选具有药用价值的新化合物合成能力的活性菌。通过抑菌实验筛选到的部分菌株对革兰氏阴性细菌(P.fluorescens)表现出较强活性,研究结果对筛选具有药用价值的新化合物合成能力的海绵来源真菌具有很好的指导意义。本研究推测PKS/NRP在海绵共生活性菌与海绵的共生关系中起到帮助宿主防御外来侵害的作用,为海绵活性物质的微生物来源假说提供了证据。
In this study, six different fungal media (Martins, 2216, M1, Gause, MYPG and Czapek Sabouraud media) and three temperature gradient (20℃、25℃、30℃) were used to investigate the phylogenetic diversity of culturable fungi associated with ten sponges collected from the South China Sea based on RFLP analysis and 18S rRNA gene sequences. Sterile oxford cup-based method was used to examine antimicrobial activities against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans. Meanwhile, PKS and NRPS genes of the 177 culturable fungi were screened by PCR method.
The results revealed that 177 fungi were isolated from 10 sponges collected from the South China Sea using 6 media at 3 different temperature, where 8 from X4, 23 from X5, 32 from X6, 1 from X7, 4 from X8, 35 from X9, 22 from X10, 17 from X11, 25 from X12 totally. There was no obvious difference among three cultivation temperature 20℃, 25℃and 30℃. Among the tested 6 media, Martins medium and MYPG medium cultivated the most fungal isolates (n=45 and n=44 respectively). In Czapek's medium and M1 medium, 32 and 24 isolates from marine sponge, respectively. Based on fungi 18S rDNA specific primers, we amplify 760bp fragments with PCR. Used Taq I, Hinf I and Hae III to accomplish RFLP enzyme-digestion differing in patterens combined with colony morphology, 24 independent fungi strains were selected. Partial sequencing of the 18S rRNA gene of these fungal isolates and analyzed by BLAST revealed that: 24 representative isolates were classified into 4 orders(Microascales、Phyllachorales、Hypocreales and Eurotiales) and 10 genera of Ascomycota. The prodominant isolates were Eurotiales (37.5%) and Hypocreales (41.6%).
All the 177 fungal isolates were examined for activity against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans.The fungal isolates showed antibacterial activity with 3(x4-167、x11-75、x12-91) against B. subtilis, 8(x6-13、x10-49、x10-63、x12-94、x12-95、x12-109、x13-105、x13-118) against P. fluorescens, especially X13-105, X12-94 and X13-118 displayed antibacterial activity against both P. fluorescens and S. aureus.
15 polyketide synthases gene sequences 790bp in length were detected in these fungal isolates. The PKS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome and Aspergillus fumigatus Af293 polyketide synthase gene by BLAST analyzed. Meanwhile, 4 nonribosomal peptides synthesis gene sequences 1200bp in length were detected in these fungal isolates. The NRPS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome by BLAST analyzed.
In this study, a great rich and diverse culturable fungi associated with sponges collected from the South China Sea was revealed by molecular identification and phylogenetic analysis.As the largest source of activity products produced by marine fungi, some strains of Penicillium and Aspergillus isolated in this study showed a broad-spectrum antimicrobial activity against bacteria.The PKS and NRPS genes were found existed in some strians,which suggested that the fungi with PKS and NRPS may play a role in sponge chemical joint defense. In the research of fungal PKS and NRPS genes, a variety of PKS or NRPS genes were found in the same strains, based on the diversity and novelty of PKS and NRPS genes which can guide the selection of bacteria with medicinal activity. Based on antibiotic activity assay, some strains showed antimicrobial activities towards P.fluorescens which had a great guidance of the screening of sponge-associated fungi with new compounds synthesis ability.Our study suggests the role of sponge-symbiosis bacteria played in the host’s chemical defense and provides further proof for the hypothesis that sponge-associated bacteria are the true producers of many novel bioactive compounds.
实验结果显示:采用6种培养基3个温度梯度的组合共分离得到177株真菌菌株,其中从X4海绵中分离到了8株真菌菌株,X5(23)、X6(32)、X7(1)、X8(4)、X9(35)、X10(22)、X11(17)、X12(25)、X13(10)。在20℃、25℃、30℃三个培养温度下分离到的真菌菌株数量没有明显差异,在所采用的6种培养基中,马丁氏培养基和MYPG培养基分离得到的菌株数最多,分别为46和44;其次是查氏和M1培养基,分别为32和24;高氏一号培养基和2216培养基再次之,分别为17和14。采用真菌18S rDNA特异引物PCR扩增,通过TaqI、HinfI、HaeIII三种限制性内切酶对PCR产物进行RFLP酶切分型,共得到24株独立菌。对24株菌的PCR产物测序并BLAST进行在线序列比对,从而得到这些菌株的生物学信息。24株菌的序列分析显示,所有分离出的真菌分别属于子囊菌门下Ascomycota 4目(小囊菌目Microascales、黑痣菌目Phyllachorales、肉座菌目Hypocreales和散囊菌目Eurotiales)10属。其中散囊菌目下的青霉属(Penicillium)和曲霉属(Aspergillus()37.5%);肉座菌目下的棒束孢属(Isaria)、拟青霉属(Simplicillium)等6个属(41.6%)是优势的真菌类群。
采用6种指标菌对分离得到的177株真菌菌株进行抑菌活性筛选,结果共有11株真菌表现出了抑菌活性,其中3株(x4-167、x11-75、x12-91)对枯草芽孢杆菌(Bacillus sp.)表现出抑菌活性,8株(x6-13、x10-49、x10-63、x12-94、x12-95、x12-109、x13-105、x13-118)对荧光假单胞菌(Pseudomonas fluorescens)表现出抑菌活性,其中x13-105, x12-94和x13-118同时对枯草芽孢杆菌(Bacillus sp.)和荧光假单胞菌(Pseudomonas fluorescens)表现出活性。获得了15条790 bp的PKS基因片段, BLAST比对结果表明这些PKS基因片段与一株产黄青霉Penicillium chrysogenum Wisconsin 54-1255的基因组序列和一株致病烟曲霉Aspergillus fumigatus Af293的PKS序列具有很高的相似性(98%-99%)。研究中筛选到的PKS基因均为I型的PKS。同时,克隆到4条1200 bp的NRPS基因片段, BLAST比对结果表明4条NRPS序列都与产黄青霉Penicilliumchrysogenum Wisconsin 54-1255的基因组序列具有很高的相似性(98%-99%)。
本研究通过分子鉴定和系统发育的分析揭示南海海绵具有非常丰富多样的可培养真菌,是丰富的海洋微生物资源。相对于其它培养基,马丁氏培养基与MYPG培养基更适合海绵真菌的分离培养。不同的海绵体内真菌组成存在差异,海绵也存在富含真菌的海绵与寡真菌海绵之分。本研究揭示了青霉和曲霉属真菌在海绵中的广泛分布。作为海洋真菌活性产物中产生最多的来源,本研究分离得到的部分青霉和曲霉菌表现出了对细菌的广谱抑菌活性,从一些菌株中发现了PKS和NRPS基因的存在,暗示我们这些带有PKS和NRPS的真菌可能在海绵化学联合防御中起作用。在对真菌PKS和NRPS研究中我们发现,在同一菌株中存在多种PKS或NRPS基因,基于PKS和NRPS基因的多样性和新颖性,可以指导筛选具有药用价值的新化合物合成能力的活性菌。通过抑菌实验筛选到的部分菌株对革兰氏阴性细菌(P.fluorescens)表现出较强活性,研究结果对筛选具有药用价值的新化合物合成能力的海绵来源真菌具有很好的指导意义。本研究推测PKS/NRP在海绵共生活性菌与海绵的共生关系中起到帮助宿主防御外来侵害的作用,为海绵活性物质的微生物来源假说提供了证据。
In this study, six different fungal media (Martins, 2216, M1, Gause, MYPG and Czapek Sabouraud media) and three temperature gradient (20℃、25℃、30℃) were used to investigate the phylogenetic diversity of culturable fungi associated with ten sponges collected from the South China Sea based on RFLP analysis and 18S rRNA gene sequences. Sterile oxford cup-based method was used to examine antimicrobial activities against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans. Meanwhile, PKS and NRPS genes of the 177 culturable fungi were screened by PCR method.
The results revealed that 177 fungi were isolated from 10 sponges collected from the South China Sea using 6 media at 3 different temperature, where 8 from X4, 23 from X5, 32 from X6, 1 from X7, 4 from X8, 35 from X9, 22 from X10, 17 from X11, 25 from X12 totally. There was no obvious difference among three cultivation temperature 20℃, 25℃and 30℃. Among the tested 6 media, Martins medium and MYPG medium cultivated the most fungal isolates (n=45 and n=44 respectively). In Czapek's medium and M1 medium, 32 and 24 isolates from marine sponge, respectively. Based on fungi 18S rDNA specific primers, we amplify 760bp fragments with PCR. Used Taq I, Hinf I and Hae III to accomplish RFLP enzyme-digestion differing in patterens combined with colony morphology, 24 independent fungi strains were selected. Partial sequencing of the 18S rRNA gene of these fungal isolates and analyzed by BLAST revealed that: 24 representative isolates were classified into 4 orders(Microascales、Phyllachorales、Hypocreales and Eurotiales) and 10 genera of Ascomycota. The prodominant isolates were Eurotiales (37.5%) and Hypocreales (41.6%).
All the 177 fungal isolates were examined for activity against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans.The fungal isolates showed antibacterial activity with 3(x4-167、x11-75、x12-91) against B. subtilis, 8(x6-13、x10-49、x10-63、x12-94、x12-95、x12-109、x13-105、x13-118) against P. fluorescens, especially X13-105, X12-94 and X13-118 displayed antibacterial activity against both P. fluorescens and S. aureus.
15 polyketide synthases gene sequences 790bp in length were detected in these fungal isolates. The PKS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome and Aspergillus fumigatus Af293 polyketide synthase gene by BLAST analyzed. Meanwhile, 4 nonribosomal peptides synthesis gene sequences 1200bp in length were detected in these fungal isolates. The NRPS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome by BLAST analyzed.
In this study, a great rich and diverse culturable fungi associated with sponges collected from the South China Sea was revealed by molecular identification and phylogenetic analysis.As the largest source of activity products produced by marine fungi, some strains of Penicillium and Aspergillus isolated in this study showed a broad-spectrum antimicrobial activity against bacteria.The PKS and NRPS genes were found existed in some strians,which suggested that the fungi with PKS and NRPS may play a role in sponge chemical joint defense. In the research of fungal PKS and NRPS genes, a variety of PKS or NRPS genes were found in the same strains, based on the diversity and novelty of PKS and NRPS genes which can guide the selection of bacteria with medicinal activity. Based on antibiotic activity assay, some strains showed antimicrobial activities towards P.fluorescens which had a great guidance of the screening of sponge-associated fungi with new compounds synthesis ability.Our study suggests the role of sponge-symbiosis bacteria played in the host’s chemical defense and provides further proof for the hypothesis that sponge-associated bacteria are the true producers of many novel bioactive compounds.
引文
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