蛹虫草聚酮合酶基因的多样性分析
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摘要
以蛹虫草的菌丝体为材料,利用基因组挖掘的方式从蛹虫草基因组中获得其聚酮合酶(Polyketide synthase,PKS)基因,对这些PKS基因进行生物信息学分析以推断它们的功能,并检测它们在不同培养基上的表达情况。结果表明:蛹虫草中含有13个PKS基因(CmPKS 1-13),包括2个非还原型聚酮合酶(Non-reducing PKS,NR-PKS),5个部分还原的聚酮合酶(Partial-reducing PKS,PR-PKS),6个高度还原型聚酮合酶(Highly reducing PKS,HR-PKS);聚类分析显示CmPKS 1、CmPKS 3可能参与链格孢吡喃酮(alternapyrone),CmPKS 4可能参与黄曲霉素,CmPKS 5可能参与美伐他汀,CmPKS 6可能参与分生孢子色素,CmPKS 8可能参与伊快霉素的生物合成,其余PKS与生物合成未知化合物的PKS聚为一支;半定量PCR显示CmPKS 7和CmPKS 9在4种培养基上均强烈表达;CmPKS 3、CmPKS 6在4种培养基上微弱表达;CmPKS 10、CmPKS 11和CmPKS 12只在GL培养基上强烈表达,在其余3种培养基上微弱表达;CmPKS 1和CmPKS 4只在GL培养上微弱表达,CmPKS 8仅在GS培养基上微弱表达;CmPKS 2、CmPKS 5和CmPKS 13在检测的培养基中都不表达。本研究为进一步异源表达蛹虫草中的PKS基因及其功能鉴定、具新颖结构聚酮化合物的发掘奠定基础。
The mycelium of Cordyceps militaris was used as the material,the present experiment has obtained some polyketide synthase(PKS) gene from the genome of C.militaris with the method of genome-mining,and carried a bioinformatics analysis of these PKS genes to perform their function,and detected these PKS gene expression in the different medium.The results showed that there were 13 PKS(CmPKS 1-13) genes in the genome of C.militaris,including 2 non-reducing PKS(NR-PKS),5 partial reducing PKS(PR-PKS),6 highly reducing PKS(HR-PKS);the phylogenetic analysis revealed CmPKS 1,CmPKS 3 could involve in the biosynthesis of alternapyrone, CmPKS 4 could involve sterigmatocystin, CmPKS 5 could involve mevastatin,CmPKS 6 could involve conidial yellow pigment, CmPKS 8 could involve equisetin,and other PKS in C.militaris that could involve some compounds were unkow;the semi-quantitative RT-PCR revealed CmPKS 7 and CmPKS 9 strongly expressed in 4 medium,CmPKS 3 and CmPKS 6 slightly expressed in all 4 medium, CmPKS 10;CmPKS 11 and CmPKS 12 only strongly expressed in GL,and slightly in other 3 medium;CmPKS 1,CmPKS 4 only expressed in GL,and CmPKS 8 only slightly expressed in GS;CmPKS 2,CmPKS 5 and CmPKS 13 not completely expressed in 4 medium.This study provides some important basis for heterologously expression of the PKS gene in C.militaris,the identification of gene function,and for finding a new putative structure of polyketide.
The mycelium of Cordyceps militaris was used as the material,the present experiment has obtained some polyketide synthase(PKS) gene from the genome of C.militaris with the method of genome-mining,and carried a bioinformatics analysis of these PKS genes to perform their function,and detected these PKS gene expression in the different medium.The results showed that there were 13 PKS(CmPKS 1-13) genes in the genome of C.militaris,including 2 non-reducing PKS(NR-PKS),5 partial reducing PKS(PR-PKS),6 highly reducing PKS(HR-PKS);the phylogenetic analysis revealed CmPKS 1,CmPKS 3 could involve in the biosynthesis of alternapyrone, CmPKS 4 could involve sterigmatocystin, CmPKS 5 could involve mevastatin,CmPKS 6 could involve conidial yellow pigment, CmPKS 8 could involve equisetin,and other PKS in C.militaris that could involve some compounds were unkow;the semi-quantitative RT-PCR revealed CmPKS 7 and CmPKS 9 strongly expressed in 4 medium,CmPKS 3 and CmPKS 6 slightly expressed in all 4 medium, CmPKS 10;CmPKS 11 and CmPKS 12 only strongly expressed in GL,and slightly in other 3 medium;CmPKS 1,CmPKS 4 only expressed in GL,and CmPKS 8 only slightly expressed in GS;CmPKS 2,CmPKS 5 and CmPKS 13 not completely expressed in 4 medium.This study provides some important basis for heterologously expression of the PKS gene in C.militaris,the identification of gene function,and for finding a new putative structure of polyketide.
引文
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[16]陈思涵,钱靖,彭杰,等.农杆菌介导的外源基因在本氏烟中瞬时表达体系优化研究[J].西南林业大学学报,2018,38(4):6-11.
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[19]Bergmann S,Schumann 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.
[20]Kirimura K,Watanabe S,Kobayashi K.Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328[J].Biochemicaland Biophysical Research Communications,2016,473(4):1106-1110.
[21]Yuan C,Guo Y H,Wang H Y, et al.Allelopathic polyketides from an endolichenic fungus Myxotrichum sp.by using OSMAC strategy[J].Scientific Reports,2016,6:19350.
[22]Kinoshita Y,Yamamoto Y,Kurokawa T, et al.Influences of nitrogen sources on usnic acid production in a cultured mycobiont of the lichen Usnea hirta (L.)Wigg[J].Journal of the Agricultural Chemical Society of Japan,2001,65(8):1900-1902.
[23]王毅,周旭,许宰铣,等.长松萝中一个聚酮合酶基因簇的克隆和鉴定[J].微生物学报,2014,54(7):770-777.
[24]李菲,王忠,卞文印,等.5种氮源对蛹虫草生长及其质量的影响[J].福建农业科技,2018(1):4-6.
[25]任昕雯,李宇轩,吴兆琦.液体发酵条件对蛹虫草产虫草素的影响[J].陕西农业科学,2018,64(4):58-60.
[2]Nag T B,Wang H.Pharmacological actions of Cordyceps,a prized folk medicine[J].Journal of Pharmacy & Pharmacology,2005,57(12):1509-1519.
[3]肖亦农,韩梅,赵春燕.不同培养基对蛹虫草子实体甘露醇、多糖和矿质元素含量的影响[J].沈阳农业大学学报,2009,40(2):227-229.
[4]曾宏彬,宋斌,李泰辉.蛹虫草研究进展及其产业化前景[J].食用菌学报,2011,18(2):70-74.
[5]王升厚,牛世莉,徐方旭,等.基于代谢组学的功能性蛹虫草成分研究[J].微生物学杂志,2018,38(2):1-7.
[6]Xiao J H,Zhong J J.Secondary metabolites from Cordyceps species and their antitumor activity studies[J].Recent Patents on Biotechnology,2007,1(2):123-137.
[7]Xia Y,Luo F,Shang Y, et al.Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin[J].Cell Chemical Biology,2017,24(12):1479-1489.
[8]陈俐彤,曹红峰,黄文芳.蛹虫草的化学成分、药效及应用[J].现代食品科技,2005,21(3):192-194.
[9]Zhang C,Ke D,Duan Y, et al.The combinatorial biosynthesis of“unnatural”products with polyketides[J].Transactions of Tianjin University,2018(15/16):1-12.
[10]Cox R J.Polyketides,proteins and genes in fungi:programmed nano-machines begin to reveal their secrets[J].Organic & Biomolecular Chemistry,2007,5(13):2010-2026.
[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):15670.
[12]董高超,刘震,莫朕,等.基因组挖掘在天然产物研究中的应用进展[J].化学与生物工程,2017,34(2):10-12.
[13]Robbel L,Knappe T A,Linne U, et al.Erythrochelin-a hydroxamate-type siderophore predicted from the genome of Saccharopolyspora erythraea[J].The Federation of European Biochemical Societies Journal,2010,277(3):663-676.
[14]K■nig C C,Scherlach K,Schroeckh V, et al.Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus[J].Organic & Biomolecular Chemistry,2013,14(8):938-942.
[15]Medema M H,Blin K,CimertmancicP, et al.AntiSMASH:rapid identification,annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences[J].Nucleic Acids Research,2011,39(S2):W339-W346.
[16]陈思涵,钱靖,彭杰,等.农杆菌介导的外源基因在本氏烟中瞬时表达体系优化研究[J].西南林业大学学报,2018,38(4):6-11.
[17]原晓龙,赵能,陈剑,等.哈茨木霉基因组中基因多样性的生物信息学分析[J].西部林业科学,2017,46(4):7-12.
[18]Zuloaga F O,Salariato D L,Scataglini A.Molecular phylogeny of Panicum s.str.(Poaceae,Panicoideae,Paniceae)and insights into its biogeography and evolution [J].Plos One,2018,13(2):e0191529.DOI:10.1371/journal.pone.0191529
[19]Bergmann S,Schumann 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.
[20]Kirimura K,Watanabe S,Kobayashi K.Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328[J].Biochemicaland Biophysical Research Communications,2016,473(4):1106-1110.
[21]Yuan C,Guo Y H,Wang H Y, et al.Allelopathic polyketides from an endolichenic fungus Myxotrichum sp.by using OSMAC strategy[J].Scientific Reports,2016,6:19350.
[22]Kinoshita Y,Yamamoto Y,Kurokawa T, et al.Influences of nitrogen sources on usnic acid production in a cultured mycobiont of the lichen Usnea hirta (L.)Wigg[J].Journal of the Agricultural Chemical Society of Japan,2001,65(8):1900-1902.
[23]王毅,周旭,许宰铣,等.长松萝中一个聚酮合酶基因簇的克隆和鉴定[J].微生物学报,2014,54(7):770-777.
[24]李菲,王忠,卞文印,等.5种氮源对蛹虫草生长及其质量的影响[J].福建农业科技,2018(1):4-6.
[25]任昕雯,李宇轩,吴兆琦.液体发酵条件对蛹虫草产虫草素的影响[J].陕西农业科学,2018,64(4):58-60.