不吸水链霉菌辽宁变种基因转移系统的建立

Establishment of Gene Transformation System for Streptomyces ahygroscopicus var.Liaoningensis

以嘧肽霉素生物合成阻断突变株UV2136为研究对象,建立了不吸水链霉菌辽宁变种的基因转移系统。UV2136对硫链丝菌素高度敏感,选择含有该抗生素标记的质粒pIJ702和pWHM3,探索其转化UV2136的可能性。结果表明:UV2136含有较强的限制修饰系统,采取热衰减法获得原生质体、原生质体热处理、冷处理、EDTA处理、质粒DNA变性等,均不能实现质粒pIJ702向UV2136的转化。来自非甲基化宿主E.coli ET12567的pWHM3也无法实现转化,而源于E.coli DH5α的甲基化pWHM3,可以低频转化菌株UV2136,转化效率分别为4转化子.μg-1 pWHM3,经自身修饰的pWHM3可实现高效转化,达到104转化子.μg-1pWHM3。

Streptomyces ahygroscopiczts var. Liaoningensis is the product of cytosinpeptidemyein, a novel antiviral antibiotic. To find its biosynthetic gene cluster, the gene transformation system was developed in this study. UV2136, a mutant blocked in cytosinpeptidemycin biosynthesis, was highly sensitive to thiostrepton, plasmid pIJ702 and pWHM3 containing thiostrepton resistance gene, tsr, were used to transform UV2136. Without endogenous plasmid, UV2136 has strong restriction and modification system which prevent the transformation of foreign plasmid. Many possible solutions were adopted, including heat attenuation, heat treatment, chill treatment, EDTA treatment and plasmid denaturation, no transformant was found when pIJ702 was used. Non-methylated pWHM3 derived from E.coli ET12567 can not be transformed into UV2136, while the pWHM3 from methylatng host, E.coli DH5α, can be introduced at a low frequency, 4 transformants per μg pWHM3, and the pWHM3 modified by UV2136 itself can be transformed at a higher frequency, 104 transformants per μg pWHM3.