Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics,such as apramycin,tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebr...Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics,such as apramycin,tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E.coli/Streptomyces shuttle vector pKC505 by in vitro packing.The probability of finding a specific gene from the library composed of 3 000 colonies was over 99 9%. According to the highly conserved sequence of the genes involved in 6\|deoxyhexose biosynthesis,primers were designed and 0 6kb fragment homologous to strE gene was obtained by PCR.30 positive clones were found from the genomic library of \%S.tenebrarius\% H6 with the 0 6kb fragment as a probe.Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions.An ORF of dTDP\|glucose\|4,6\|dehydratase gene consisted of 1,132bp,designated as apr E,was obtained and submitted to GenBank under the accession number of AF306787.A DNA sequence highly homologous to str L coding dTDP\|4\|dehydrorhamnose reductase was found linked with apr E gene.展开更多
文摘S.tenebrarius H6主要产生安普霉素、氨甲酰妥布霉素和氨甲酰卡那霉素B,其中后两者仅在3′位有差异。为克隆相关基因,根据已报道的aprD3、livY、gentY基因设计兼并性PCR引物,并采用SON-PCR(singleoligonucleotide nested PCR)方法和LASP-PCR(linear amplification single primer PCR)方法,扩增获得部分SCD1基因,Blast分析为短链脱氢酶基因。采用基因阻断技术,使S.tenebrariusH6中SCD1基因失活。与野生菌株相比,变株的抗生素组分和含量几乎没有变化,但变株产孢子能力下降,且产孢子时间推迟,推测该基因与孢子的生成调节相关。
文摘Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics,such as apramycin,tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E.coli/Streptomyces shuttle vector pKC505 by in vitro packing.The probability of finding a specific gene from the library composed of 3 000 colonies was over 99 9%. According to the highly conserved sequence of the genes involved in 6\|deoxyhexose biosynthesis,primers were designed and 0 6kb fragment homologous to strE gene was obtained by PCR.30 positive clones were found from the genomic library of \%S.tenebrarius\% H6 with the 0 6kb fragment as a probe.Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions.An ORF of dTDP\|glucose\|4,6\|dehydratase gene consisted of 1,132bp,designated as apr E,was obtained and submitted to GenBank under the accession number of AF306787.A DNA sequence highly homologous to str L coding dTDP\|4\|dehydrorhamnose reductase was found linked with apr E gene.
