【作者】 周选围； 王子楠； 魏雅敏； 蒋科技； 尹燡周； 林娟； 孙小芬； 唐克轩；

【Author】 Zhou Xuanwei~1 Zinan Wang~2 Keji Jiang~2 Wei Yamin~2 Lin Juan~2 Sun Xiaofen~2 Kexuan~(1,2*) 1 Plant Biotechnology Research Center,School of Agriculture and Biology,Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,Shanghai Jiao Tong University,Shanghai,200030;2 State Key Laboratory of Genetic Engineering,School of Life Seiances,Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,Fudan University,Shanghai,200433

【机构】 上海交通大学农业与生物学院植物生物技术研发中心； 复旦大学生命科学学院遗传工程国家重点实验室；

【摘要】 采用常规分离方法,从南方红豆杉(Taxus chinensis var.mairei)中分离出内生真菌38株。根据红豆杉紫杉醇代谢途径中关键酶—紫杉二烯合成酶(Taxadiene synthase,TXS)基因的保守序列合成引物TsF(5′-CAAACCCATGTCGAATTGAGAAG-3′)和TsR(5′-AGATTCCAGA GTGTATGCAAACTTG-3′),对分离出的内生真菌DNA 进行PCR 扩增,结果表明,从38株内生真菌中的12株中能扩增出TXS 的核心片断。然后对PCR 阳性的目标菌株进行液体培养,用HPLC-MS 等方法对培养物中紫杉醇和其它紫杉烷类化合物的含量和分子量进行了进一步研究。结果发现,其中3株能够产生紫杉醇和其它紫杉烷类化合物。该研究说明,TS 基因的分子检测可作为初筛紫杉醇产生菌的有效方法,这一改进有效地提高了筛选紫杉醇产生菌的工作效率。更多还原

【Abstract】 A total of 38 endophytic fungus strains were isolated from Taxus chinensis var.mairei by aseptictechnique.Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reac-tion(PCR)analysis using primers TsF(5’-CAAACCCATGTCGAATTGAGAAG-3’)and TsR primer(5’-A-GATTCCAGAGTGTATG-CAAACTTG-3’)designed and synthesized based on the conserved sequence of TaxusTaxadiene synthase(TXS)genes.Twelve out of 38 isolated endophytic fungus strains showed PCR positive forthe TXS gene.Subsequently,taxol and taxol-related compounds were extracted from culture filtrates and myceliaof these PCR positive strains,separated by thin layer chromatography and analyzed by high performance liquidchromatography-mass spectrum(HPLC-MS).The analysis result showed that 3 strains could produce taxol andtaxol-related compounds.This study indicates that that molecular detection of the TXS gene is an efficientmethod for primary screening of taxol or taxol-related compounds-producing endophytic fungi which can im-prove effectively screening efficiency.更多还原