基于NRPS基因筛选与鉴定葡萄附生真菌
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摘要
以非核糖体多肽合成酶(nonribosomal peptide synthetase,NRPS)基因为筛选靶点,从葡萄中筛选出含有NRPS基因的附生真菌,以期发现具有抗菌作用的环肽类化合物。采用平板划线技术、斜面分离纯化技术分离葡萄附生真菌,从中筛选出一株含有NRPS基因的附生真菌(编号PTLM-1)。在此基础上,构建系统发育进化树,结合形态学方法,将该菌株鉴定为腐皮镰刀菌Fusarium solani。经过NRPS系统发育进化树分析,发现该菌株有产生抗菌环肽类化合物——环孢菌素的潜力。通过对该菌的发酵产物进行电喷雾质谱分析,进一步证实了该预测结果。该研究为定向筛选产生环肽类化合物的菌株提供了新的研究方法与理论依据。
Targeting a nonribosomal peptide synthetase (NRPS) gene, an epiphytic fungi containing NRPS gene was screened from grapes so as to discover cyclic peptide compounds with antimicrobial activities. An epiphytic fungi with NRPS gene, which named as PTLM-1, was isolated from grapes by streak plate and slant culture methods. The strain was identified as Fusarium solani by phylogenetic tree analysis and morphological observation. It was found that the strain had the potential to produce a cyclic peptide cyclosporine based on NRPS phylogenetic analysis, which was further confirmed by the electrospray ionizsation mass spectrum (ESI-MS) analysis. This study provides a new research method and theoretical basis for target-screening strains producing cyclic peptides.
Targeting a nonribosomal peptide synthetase (NRPS) gene, an epiphytic fungi containing NRPS gene was screened from grapes so as to discover cyclic peptide compounds with antimicrobial activities. An epiphytic fungi with NRPS gene, which named as PTLM-1, was isolated from grapes by streak plate and slant culture methods. The strain was identified as Fusarium solani by phylogenetic tree analysis and morphological observation. It was found that the strain had the potential to produce a cyclic peptide cyclosporine based on NRPS phylogenetic analysis, which was further confirmed by the electrospray ionizsation mass spectrum (ESI-MS) analysis. This study provides a new research method and theoretical basis for target-screening strains producing cyclic peptides.
引文
[1]王伟,朱平,程克棣.药用植物基因组及EST研究[J].中国生物工程杂志,2004,5(1):1-5.
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[16]魏景超.真菌分类鉴定手册[M].上海:上海科技出版社,1979:487-533.
[17]张梅,徐良雄,薛璟花,等.一株茄病镰刀菌的代谢产物研究[J].热带亚热带植物学报,2012,36(6):585-590.
[18]Fukai S,Tanimoto S,Maeda A,et al.Pharmacological activity of compounds extracted from persimmon peel(Diospyros kaki THUNB.)[J].J Oleo Sci,2009,58(4):213-219.
[19]刘怡君,潘沐,王明洋,等.海芦笋内生真菌基于PKSⅠ型功能基因的分离与鉴定[J].食品科学,2014,72(11):115-119.
[20]于志伟,杨欣,曾宪军,等.腐皮镰刀菌R13发酵产大黄素的初步研究[J].四川大学学报(自然科学版),2015,52(5):1135-1140.
[21]方金瑞,唐信东,任林英,等.氨基酸对环孢菌素A生物合成的影响[J].中国抗生素杂志,1990,12(2):140-141.
[22]方剑英.环孢菌素A衍生物的合成及其生物学活性研究[D].福州:福建医科大学,2008.
[23]温耀明.环孢菌素H衍生物的合成及其生物学活性研究[D].福州:福建医科大学,2007.
[24]陈振伟.茄病镰刀菌产生的环孢菌素L及其未知组分的研究[D].福州:福建医科大学,2007.
[25]陈秀明.茄病镰刀菌产生的环孢菌素小组分的研究[D].福州:福建医科大学,2008.
[2]许静,徐俊.海洋共附生微生物天然产物生物合成基因研究进展[J].微生物学报,2008,219(7):975-979.
[3]朱鹏.珍珠膜海绵共附生微生物PKS与NRPS的筛选与模块结构研究[D].杭州:浙江大学,2008.
[4]Moore B S.Biosynthesis of marine natural products:microorganisms(Part A)[J].Nat Prod Rep,2005,22(5):580-593.
[5]文艳苹.基于PKS、NRPS基因的抗生素paenimacrolidin和嗜铁素paenibactin研究[D].杭州:浙江大学,2011.
[6]朱孟沼,崔晓龙,李铭刚,等.发现微生物药物的新途径:从基因到新化合物[J].天然产物研究与开发,2006,12(5):854-857.
[7]张薇.海绵共附生活性菌PKS和NRPS基因的筛选及多样性研究[D].上海:上海交通大学,2008.
[8]符浩.基于放线菌基因组Ⅰ型聚酮合酶和非核糖体肽合成酶保守序列筛选天然活性化合物的研究[D].上海:华东理工大学,2012.
[9]Chang Z,Flatt P,Gerwick W H,et al.The barbamide biosynthetic gene cluster:a novel marine cyanobacterial system of mixed polyketide synthase(PKS)-non-ribosomal peptide synthetase(NRPS)origin involving an unusual trichloroleucyl starter unit[J].Gene,2002,296(1-2):235-247.
[10]肖吉,张光涛,朱义广,等.海洋微生物次级代谢产物生物合成的研究进展[J].中国抗生素杂志,2012,34(4):241-253.
[11]Marahiel M A,Stachelhaus T,Mootz H D.Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis[J].Chem Rev,1997,97(7):2651-2674.
[12]Liang X H,Cai Y J,Liao X R,et al.Isolation and identification of a new hypocrellin A-producing strain Shiraia sp.SUPER-H168[J].Microbiol Res,2009,164(1):9-17.
[13]燕勇,李卫平,高雯洁,等.r DNA-ITS序列分析在真菌鉴定中的应用[J].中国卫生检验杂志,2008,56(10):1958-1961.
[14]Kurtzman C P,Robnett C J.Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit(26S)ribosomal DNA partial sequences[J].Antonie Van Leeuwenhoek,1998,73(4):331-371.
[15]O’Donnell K,Cigelnik E.Two divergent intragenomic r DNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J].Mol Phylogenet Evol,1997,7(1):103-116.
[16]魏景超.真菌分类鉴定手册[M].上海:上海科技出版社,1979:487-533.
[17]张梅,徐良雄,薛璟花,等.一株茄病镰刀菌的代谢产物研究[J].热带亚热带植物学报,2012,36(6):585-590.
[18]Fukai S,Tanimoto S,Maeda A,et al.Pharmacological activity of compounds extracted from persimmon peel(Diospyros kaki THUNB.)[J].J Oleo Sci,2009,58(4):213-219.
[19]刘怡君,潘沐,王明洋,等.海芦笋内生真菌基于PKSⅠ型功能基因的分离与鉴定[J].食品科学,2014,72(11):115-119.
[20]于志伟,杨欣,曾宪军,等.腐皮镰刀菌R13发酵产大黄素的初步研究[J].四川大学学报(自然科学版),2015,52(5):1135-1140.
[21]方金瑞,唐信东,任林英,等.氨基酸对环孢菌素A生物合成的影响[J].中国抗生素杂志,1990,12(2):140-141.
[22]方剑英.环孢菌素A衍生物的合成及其生物学活性研究[D].福州:福建医科大学,2008.
[23]温耀明.环孢菌素H衍生物的合成及其生物学活性研究[D].福州:福建医科大学,2007.
[24]陈振伟.茄病镰刀菌产生的环孢菌素L及其未知组分的研究[D].福州:福建医科大学,2007.
[25]陈秀明.茄病镰刀菌产生的环孢菌素小组分的研究[D].福州:福建医科大学,2008.