产生芳香环聚酮类天然产物放线菌的分子筛选研究

Molecular screening and distribution of aromatic polyketide antibiotics producers from Actinomycetes

目的建立一套简便、快速、高效的芳香环聚酮类化合物产生菌基因筛选体系,为聚酮类药物筛选搭建一个新的技术平台;认识PKS-II基因在不同环境放线菌群中的分布规律,为新药筛选中微生物资源的定向分离提供依据。方法通过对芳香环聚酮类化合物生物合成途径所用的II型聚酮合酶氨基酸和核苷酸序列的同源性比较分析,设计一对简并引物且以微波炉法提取的基因组DNA为模板扩增KS/CLF功能域约0.8kb目的基因片段,依据放线菌基因组中II型PKS合酶基因的存在与否标定可能的芳香环聚酮类天然产物产生菌。结果利用建立的芳香环聚酮类化合物产生菌基因筛选体系对99株嗜碱放线菌、100株链霉菌、47株嗜盐放线菌和776株一般放线菌进行了筛选,研究表明链霉菌、嗜碱放线菌、嗜盐放线菌和一般放线菌II型聚酮合酶基因的阳性率分别为54%、29.3%、25.5%和24.1%。结论组合放线菌基因组DNA快速提取技术和PKSII基因简并引物PCR扩增技术建立了快速、简便、高效的芳香环聚酮类化合物产生菌基因筛选体系并对不同环境条件的放线菌菌群的PKSII基因分布进行了研究。结果表明放线菌PKSII聚酮合酶分布具有广泛性;而且同时表明链霉菌是芳香环聚酮类化合物的最丰富来源;在极端环境放线菌中,嗜盐放线菌的PKSII基因在中度嗜盐放线菌分布的频率远高?

Objective To establish practical and effective aromatic polyketide (PKS II) gene screening system and investigate the distribution of producing strain in Actinomycetes. Method A pair of degenerate PCR primers, which are located at KS and CLF gene, respectively, was designed and synthesized. Biomass was picked up from slant, and genomic DNA was extracted by microwave DNA extracting method. The DNA was used as template for PCR amplification. PCR product was analyzed by gel electrophoresis. The positive strain was distinguished by 0.8kb objective DNA band overlapping from KS to CLF regions. Result By PKS-II gene screening system 1022 strains have been investigated, including 99 alkalophilic actinomycete strains, 47 halophilic actinomycete strains, 100 streptomycete strains, and 776 other actinomycete strains. Results showed that 29.3% is positive in alkalophilic actinomycete strains, 54% is positive in Streptomyces, 25.5% and 24.1% is positive in halophilic actinomycete strains and other actinomycete strains, respectively. Conclusion The PKS-II gene was distributed in microorganisms isolated from extreme and common environment widely. Streptomyces is the richest resources for aromatic polyketide compound. This study also showed that the extreme environment microorganism, especially the alkalophilic actinomycete and the halotolerant actinomycete, is also the important resources for bioactive compounds.