| Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics, such as Apramycin, Tobramycin and Kanamycin B, etc. To study the antibiotic biosynthesis gene, the genomic library from the S. tenebrarius H6 was established firstly. S. tenebrarius H6 total DNA was extracted and incomplete digested with Sau3A I . 20?Okb fragment was segregated by sucrose gradient sedimentation and cloned into pKC5 05, Escherichia. co/il Strep!omyces shuttle vector. After in vitro packing, the recombinant vector were transduct into F. co/i DH1 competent cell. 2,880 transductants were selected. The probability of finding a specific gene from the library was over99.9%.To find a proper probe, StrE, StrD, StrQ from Streptomycin-producing strain Streptomyces griseus were used to probe S. tenebrariiis H6 total DNA, but no positive signal was found. After alignment analysis of 14 highly homologous dTDP-glucose-4,6-dehygratases, two primers were designed according to the highest conservative sequence and 0.6kb fragment from S. tenebrarius H6 homologous to strE was obtained by PCR.After using the 0.6kb fragment probe genomic library of S. tenebrarius H6, 30 positive clones were obtained. Overlapped regions were localized by southern hybridization and putative sugar related biosynthetic gene cluster which about 50kb was mapped by restriction enzyme digestions. An ORF of dTDP-glucose-4,6-dehygratase gene consisted of 1,1 32bp designated as aprE was obtained and submitted to GenBank, under the accession number of3AF306787. A DNA sequence highly homologous to strL(STRGR) coding dTDP-4-dehydrorhamnose reductase was found linked with aprE.Three S. lividans TK24 transformants of different cosmids whose insertion overlapped all 50kb gene were obtained, but no bioactivty was observed after mix culture detection.
|
PDF Full Text Request