Tn4560在圈卷产色链霉菌中的转座及其在尼可霉素生物合成相关基因克隆中的应用

采用常规转化方法用来自天蓝色链霉菌J1 5 0 1的质粒pUC1 1 6 9(pMT6 6 0∷Tn45 5 6∷vph)多次转化尼可霉素产生菌圈卷产色链霉菌野生型 71 0 0的原生质体 ,均未得到转化子。采用限制性热衰减法于 5 0℃ ,3 0min溶菌制备 71 0 0的原生质体 ,获得了转化子 ,但转化频率极低 ,只有 0 4个转化子 μgDNA。用来自 71 0 0的pUC1 1 6 9再转化不含pUC1 1 6 9的 71 0 0原生质体 ,转化频率提高 1 0 3 ～ 1 0 4 倍。于 3 9℃ ,MM Vio条件下培养携带有pUC1 1 6 9的 71 0 0孢子 ,Tn45 6 0发生转座 ,筛选到 40 6 8个转座菌落 ,并从中得到 8株尼可霉素阻断突变株 ;对这 8株突变株的总DNA进行Southern杂交分析表明 ,Tn45 6 0至少在 4个不同的位点插入到 71 0 0的染色体上。用实验室已获得的与尼可霉素生物合成有关的 3 0kbDNA片段为探针和经不同酶切的 8株突变株的总DNA进行Southern杂交 ,结果表明 ,除阻断突变株Nik5有杂交信号且杂交信号大小均同野生型…

Tn4560 Transposition in Streptomyces ansochromogenes and Its Application for Cloning of Genes Involved in Nikkomycin Biosynthesis

No transformant was obtained when pUC1169(pMT660::Tn4556::vph) from S. coelicolor J1501 was used to transform the protoplast of S. ansochromogenes by general transformation for many times. Transformants were obtained when the protoplast of S. ansochromogenes was prepared at 50 degrees C for 30 min by heat attenuation of restriction. But the transformation frequency is very low, only 0.4 transformant per microgram DNA. Transformation frequency increased 10(3)-10(4) times when the protoplast of S. ansochromogenes was transformed by pUC1169 from itself. With Tn4560 transposition, 8 mutants lost ability to produce nikkomycin were screened from 4068 colonies with VioR-ThioS. Southern blotting suggested that Tn4560 was inserted in at least 4 positions of chromosomal DNA in S. ansochromogenes. By using the cloned 30 kb DNA fragment involved in nikkomycin biosynthesis as a probe, southern hybridization was carried out with 8 mutant total DNA digested with different enzymes. Only total DNA from Nik5 displayed signals, which were the same as those of the wild type. With HPLC analysis, 8 mutants could be classified into two types for nikkomycin production, one is Nik5, the other is Nik1, 2, 3, 4, 6, 7 and Nik8. As deduced preliminarily, the deletion of large chromosomal DNA fragment involved in nikkomycin biosynthesis took place in the 7 mutants. The partial gene library of Nik5 was constructed, the 4.2 kb and the 0.6 kb chromosomal DNA fragment flanking Tn4560 were cloned. Sequence analysis showed that the 4.8 kb DNA fragment (GenBank accession number:AF349618) contains two complete ORFs and one partial ORF.