平板影印与AHBA合成酶基因筛选用于安莎类抗生素产生菌的分离  

Replica Plating and Gene Screening Combined to Separate AHBA Synthase Gene Positive Actinomycetes

作　　者：徐祯[1] 徐庆妍[1] 胡志钰[1] 郑忠辉[1] 黄耀坚[1] 

机构地区：[1]厦门大学生命科学学院,福建厦门361005

出　　处：《厦门大学学报（自然科学版）》2012年第1期107-111,共5页Journal of Xiamen University：Natural Science

基　　金：中央高校基本业务费资助项目(2010121092;2011121037);科技部国际科技合作项目(2007DFA30970)

摘　　要：基因筛选是筛选具有抗生素产生潜力微生物的新方法.针对筛选中单菌落分离纯化不仅工作量大,而且耗费时间,采用一种将平板影印技术与PCR扩增技术相结合从土壤中快速获得具有安莎类抗生素产生潜力的放线菌的方法.根据安莎类抗生素合成途径中的3-氨基-5-羟基苯甲酸合成酶(AHBAs)基因的保守性,通过放线菌的培养、影印、AH-BAs基因的PCR扩增,已从33份土样中获得8株AHBAs阳性菌株.结果表明:该方法是一种非常有效的能够快速获得AHBAs基因阳性菌株的方法,并且该方法可扩展用于从土壤中分离其他有价值的抗生素产生菌.Gene mining is a new strategy to discover microbial natural products. However,it is laborious to inoculate and purify test- ed strains before gene amplification, so gene mining can not be applied to large amounts of samples. Herein, a high throughput method for the gene mining of soil actinomycetes, combining the replica plating technique and PCR amplification was recommended. In the ＂present work,3-amino-5-hydroxybenxoic acid synthase （AHBAs） were used as amplicated gene. Through soil actinomyctes culture, colony replica planting, PCR amplification of AHBAs genes, isolation of positive strains and characterization of fermentation extracts, eight AHBAs positive strains were selected from 33 plants rhizosphere soil samples. The results showed that the replica plating-gene screening combined technique was useful for the rapid selection of ansamycin-producing actinomycetes, and it could be applied to search for producers of other antibiotics in natural soils.

关 键 词：放线菌 3-氨基-5-羟基苯甲酸合成酶基因 高通量筛选 PCR扩增 平板影印 

分 类 号：Q935[生物学—微生物学]