红色糖多孢菌染色体基因快速失活技术研究及应用

目的：研究红色糖多孢菌染色体快速同源重组基因失活技术。方法：以Thio抗性基因（tsr）作为筛选标记,在其上下游分别连接失活目的基因两侧同源片段,利用PEG介导原生质体转化将线性DNA同源片段转入红色糖多孢菌,通过tsr基因两侧同源片段与染色体同时重组,将目的基因敲除或失活。结果：tsr基因两侧同源片段长度与红色糖多孢菌染色体同源重组效率密切相关,同源片段长度在500 bp左右时,线性片段与染色体有效重组率很低,而同源片段长度超过1000 bp时,可获得有效的基因失活突变菌株。通过这种技术构建的红色糖多孢菌ΔbldD基因突变体,合成红霉素产量显著降低,说明bldD参与红霉素合成基因调控。结论：红色糖多孢菌染色体快速同源重组基因失活技术,对于分析红色糖多孢菌基因功能具有重要作用。

Rapid chromosomic gene-inactivating technology of Saccharopolyspora erythraea

Objective： To explore the rapid chromosomic gene-inactivating technology of Saccharopolyspora erythraea. Methods：Linear DNA containing thiostrepton resistance gene （tsr） and upper and down homologous fragments of the target inactivated gene around tsr was transformed into S. erythraea under PEG mediation, and by chromosomic homologous recom- bination, the mutant in which the target gene had been disrupted could be screened out with thiostrepton choice. Results： The recombinant rate was related to the length of homologous fragments, and 500 bp-length homologous fragments could hardly recombine with chromosome of the bacteria, while fragments longer than 1 000 bp worked well. With this method, S. erythraea mutants with both KR6 and bldD genes inactivated were obtained. Conclusion：A rapid gene-inactivating tech- nology is established by chromosomic homologous recombination, and it plays an important role in analysis of the gene func- tion of S. erythraea.