皮革肾岛衣一个聚酮合酶基因的克隆与鉴定
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摘要
通过对地衣型真菌皮革肾岛衣转录组数据的分析,挖掘出1条非还原型聚酮合酶基因,通过RT-PCR首次克隆得到该基因的cDNA全长(NpPKS2),并通过生物信息学手段分析其基因和蛋白氨基酸序列,采用荧光定量PCR技术分析该基因在不同培养基上的表达情况.结果显示:NpPKS2基因全长6 249 bp,编码2 082个氨基酸;生物信息学分析显示该基因编码1种非还原型聚酮合酶,结构域顺序为SAT-KS-AT-PT-ACP-TE,根据聚类分析和结构域分析,推断NpPKS2为苔色酸合成酶;荧光定量分析显示地衣型真菌皮革肾岛衣中的NpPKS2基因用BMG培养基培养时表达量最高.
A non-reducing polyketide synthase(NR-PKS) gene was excavated from the transcriptome of lichen-forming fungus Nephrmopsis pallescens, Its full-length cDNA(NpPKS2) was obtained by RT-PCR, and followed by sequencing its DNA and proteinic amino acids sequence by bioinformatics. Lastly, expressions of NpPKS2 gene in different medium were compared using real-time fluorescence quantitative PCR. The results showed that the full-length of NpPKS2 gene was 6 249 bp, which encoded 2 082 amino acids. Bioinformatics analysis unveiled that the NpPKS2 gene encoded a NR-PKS enzyme, with its domain being SAT-KS-AT-PT-ACP-TE sequentially, and could infer that NpPKS2 might be orsellinic acid synthase(OAS) according to the phylogenetic tree and domain structure. Furthermore, realtime fluorescence quantitative PCR revealed that the expression level of NpPKS2 gene peaked when the N.pallescens LFF was cultivated in media BMG.
A non-reducing polyketide synthase(NR-PKS) gene was excavated from the transcriptome of lichen-forming fungus Nephrmopsis pallescens, Its full-length cDNA(NpPKS2) was obtained by RT-PCR, and followed by sequencing its DNA and proteinic amino acids sequence by bioinformatics. Lastly, expressions of NpPKS2 gene in different medium were compared using real-time fluorescence quantitative PCR. The results showed that the full-length of NpPKS2 gene was 6 249 bp, which encoded 2 082 amino acids. Bioinformatics analysis unveiled that the NpPKS2 gene encoded a NR-PKS enzyme, with its domain being SAT-KS-AT-PT-ACP-TE sequentially, and could infer that NpPKS2 might be orsellinic acid synthase(OAS) according to the phylogenetic tree and domain structure. Furthermore, realtime fluorescence quantitative PCR revealed that the expression level of NpPKS2 gene peaked when the N.pallescens LFF was cultivated in media BMG.
引文
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[23] J?RGENSEN S H, FRANDSEN R J, NIELSEN K F, et al. Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis[J]. Fungal Genetics & Biology, 2014,70(6):24-31.
[24] ZHOU H, ZHAN J, WATANABE K, et al. A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(17):6 249-6 254.
[25] SHIMIZU T, KINOSHITA H, ISHIHARA S, et al. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus[J]. Applied Environmental Microbiology, 2005,71(7):3 453-3 457.
[26] SCHüMANN J, HERTWECK C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes[J]. Journal of Biotechnology, 2006,124(4):690-703.
[27] BERGMANN S, SCHüMANN J, SCHERLACH K, et al. Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans[J]. Nature Chemical Biology, 2007,3(4):213-217.
[28] YUAN C, GUO Y H, WANG H Y, et al. Allelopathic polyketides from an endolichenic fungus Myxotrichum sp. by using OSMAC strategy[J/OL]. Scientific reports, 2016,6:19 350.DOI: 10.1038/srep19350.
[2] LI Y, XU W, TANG Y. Classification, prediction, and verification of the regioselectivity of fungal polyketide synthase product template domains[J]. Journal of Biological Chemistry, 2010,285(30):22 764-22 773.
[3] GATTO G J, BOYNE M T, KELLEHER N L, et al. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster[J]. Journal of the American Chemical Society, 2006,128(11):3 838-3 847.
[4] K?ADNA A, MICHALSKA T, BERCZY■, et al. Evaluation of the antioxidant activity of tetracycline antibiotics in vitro[J]. Luminescence, 2012,27(4):249-255.
[5] MALAIYANDI V, HOULE M C, SKOTNICHIGRANT S. Airborne allergic contact dermatitis from tylosin in pharmacy compounders and cross-sensitization to macrolide antibiotics[J]. Dermatitis Contact Atopic Occupational Drug, 2012,23(5):227-230.
[6] HERTWECK C. The biosynthetic logic of polyketide diversity[J]. Angewandte Chemie International Edition, 2009,48(26):4 688-4 716.
[7] STARCEVIC A, WOLF K, DIMINIC J, et al. Recombinatorial biosynthesis of polyketides[J]. Journal of Industrial Microbiology & Biotechnology, 2012,3(3):503-511.
[8] COX R J. Polyketides, proteins and genes in fungi: programmed nano-machines begin to reveal their secrets[J]. Organic & biomolecular chemistry, 2007,5(13):2 010-2 026.
[9] SANCHEZ J F, CHIANG Y M, SZEWCZYK E, et al. Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans[J]. Molecular Biosystems, 2010,6(3):587-593.
[10] WILLIAMS R B, HENRIKSON J C, HOOVER A R, et al. Epigenetic remodeling of the fungal secondary metabolome[J]. Organic & Biomolecular Chemistry, 2008,6(11):1 895-1 897.
[11] KROKEN S, GLASS N L, TAYLOR J W, et al. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003,100(26):15 670-15 675.
[12] WANG Y, KIM J A, CHEONG Y H, et al. Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima[J]. Mycological Progress, 2011,11(1):75-83.
[13] SHEN B. Polyketide biosynthesis beyond the typeⅠ, Ⅱ and Ⅲ polyketide synthase paradigms[J]. Current Opinion in Chemical Biology, 2003,7(2):285-295.
[14] FULTON T R, IBRAHIM N, LOSADA M C, et al. A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium[J]. Molecular and General Genetics, 1992,262(4-5):714-720.
[15] FAILLE A, GAVALDA S, SLAMA N, et al. Insights into substrate modification by dehydratases from type I polyketide synthases[J]. Journal of Molecular Biology, 2017,429(10):1 554-1 569.
[16] WATANABE A, EBIZUKA Y. Unprecedented mechanism of chain length determination in fungal aromatic polyketide synthases[J]. Chemistry & Biology, 2004,11(8):1 101-1 106.
[17] SATTELY E S, FISCHBACH M A, WALSH C T. Total biosynthesis: in vitro reconstitution of polyketide and nonribosomal peptide pathways[J]. Natural Product Reports, 2008,39(46):757-793.
[18] TSAI H F, CHANG Y C, WASHBURN R G, et al. The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence[J]. Journal of Bacteriology, 1998,180(12):3 031-3 038.
[19] MAYORGA M E, TIMBERLAKE W E. The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases[J]. Molecular & General Genetics Mgg, 1992,235(2-3):205-212.
[20] ZHANG A, LU P, DAHL-ROSHAK A M, et al. Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis[J]. Molecular Genetics and Genomics, 2003,268(5):645-655.
[21] SANCHEZ J F, CHIANG Y M, SZEWCZYK E, et al. Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans[J]. Molecular Biosystems, 2010,6(3):587-593.
[22] SCHROECKH V, SCHERLACH K, NUTZMANN H W, et al. Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009,106(34):14 558-14 563.
[23] J?RGENSEN S H, FRANDSEN R J, NIELSEN K F, et al. Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis[J]. Fungal Genetics & Biology, 2014,70(6):24-31.
[24] ZHOU H, ZHAN J, WATANABE K, et al. A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(17):6 249-6 254.
[25] SHIMIZU T, KINOSHITA H, ISHIHARA S, et al. Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus[J]. Applied Environmental Microbiology, 2005,71(7):3 453-3 457.
[26] SCHüMANN J, HERTWECK C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes[J]. Journal of Biotechnology, 2006,124(4):690-703.
[27] BERGMANN S, SCHüMANN J, SCHERLACH K, et al. Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans[J]. Nature Chemical Biology, 2007,3(4):213-217.
[28] YUAN C, GUO Y H, WANG H Y, et al. Allelopathic polyketides from an endolichenic fungus Myxotrichum sp. by using OSMAC strategy[J/OL]. Scientific reports, 2016,6:19 350.DOI: 10.1038/srep19350.