产生大环聚酮类天然产物放线菌的分子筛选研究

目的 建立一套简便、快速、高效的大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—I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 通过对大环聚酮类化合物生物合成途径所用的I型聚酮合成酶氨基酸和核苷酸序列的同源性比较分析，设计了一对引物用于扩增KS／AT功能域约1．6kb目的基因片段，依据放线菌基因组中I型PKS合成酶基因的存在与否标定可能的大环聚酮类天然产物产生菌。结果 利用建立的大环聚酮类化合物产生菌基因筛选体系对98株嗜碱放线菌、99株链霉菌、46株嗜盐放线菌和757株稀有放线菌进行了筛选，研究表明嗜碱放线菌、链霉菌、嗜盐放线菌和稀有放线菌I型聚酮合成酶基因的阳性率分别为26．5％、20．4％、15．2％和9．35％。结论 利用组合放线菌基因组DNA快速提取技术和PKS—I基因兼并引物PCR扩增技术建立了快速、简便、高效的大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PKS—I基因分布进行了研究，结果 不仅表明了放线菌PKS I聚酮合成酶分布的广泛性，而且表明极端环境放线菌，尤其是嗜碱放线菌是大环聚酮类化合物潜在的丰富资源，为新药筛选有效菌源的确定提供了可靠依据。

Molecular screening and distribution of polyketide antibiotics producers from Actinomycetes

Objective To establish a series of simple, rapid and highly efficient screening system and to investigate the distribution of the producing strain of complex polyketide antibiotics. Method A pair of degenerate PCR primers, which are located at KS and AT 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 1.6kb objective DNA band overlaped from KS to AT regions. Result A rapid, simple and effective PKS-I gene screening system was established. 1002 strains have been investigated by the method, 98 alkaliphlic actinomycete strains, 46 halophilic actinomycete strains, 99 streptomycete strains, and 759 other actinomycete strains were screened. Results showed that 26.5% was positive in alkaliphilic actinomycete strains, 20.4% was positive in Streptomyces spp., 15.2% and 9.35% were positive in halophilic actinomycete strains and other actinomycete strains, respectively. Conclusion The PKS-I gene was widely distributed in microorganisms isolated from extreme and common environment. The research also showed that the extreme environment microorganism, especially the alkaliphilic actinomycete, is the important resource for bioactive compounds.