黑暗链霉菌DNA转移系统的建立

研究了黑暗链霉菌的基因转移系统，探索了通过PEG介导的原生质体转化、接合转移向黑暗链霉菌中转入外源DNA的可能性。多次尝试用质粒pIJ702转化黑暗链霉菌9904原生质体均未成功。对原生质体进行“热处理”后转化、利用单链DNA转化等都不能将质粒导入黑暗链霉菌中，表明黑暗链霉菌对外源DNA有很强的限制修饰作用。利用接合转移将具有oriT的大肠杆菌链霉菌穿梭质粒pHZ132转入大肠杆菌ET12567(pUZ8002)中，获得供体菌ET12567(pUZ8002，pHZ132)。将供体菌与预萌发的黑暗链霉菌9904的孢子进行接合转移，成功地将pHZ132转入黑暗链霉菌9904中。质粒pHZ132经黑暗链霉菌自身修饰后也可转入黑暗链霉菌9904菌株的原生质体中，转化率约为103/μg DNA(pHZ132)。

Construction of DNA Transfer System of Streptomyces tenebrarium

To establish a gene transfer ssystem in Streptomyces tenebrarius, several methods including PEG-mediated transformation of protoplasts, conjugal transfer were investigated. Many attempts were made to introduce plasmid pIJ702 into Sreptomyces tenebrarius. It was found that plasmid pIJ702 isolated from S. lividans TK24 failed to transform the protoplasts of Sreptomyces tenebrarius. No transformant was achieved even if the protoplast was inactivated by heat treatment or dsDNA was converted ssDNA before transformation. All the results suggested that Sreptomyces tenebrarius exists a strong restriction and modification system for the transformation of foreign DNA. A recombinant E. coli ET12567 (pUZ8002, pHZ132) was obtained by transforming E. coli ET12567(pUZ8002) with oriT-containing E. coli-Sreptomyces shuttle plasmid pHZ132. In mating experiments, E. coli ET12567 (pUZ8002, pHZ132) was the donor, and the recipient was Sreptomyces tenebrarius 9904 spores after pregerminating by heat shock. Matings between dornor and recipient were conducted. Plasmid pHZ132 was introduced into Sreptomyces tenebrarius 9904 by conjugation from E. coli ET12567. The transfer system of Sreptomyces tenebrariu was established by conjugation. S. tenebrarius 9904 protoplasts were transformed by plasmid DNA modified by the host itself, and the transformation frequency was about 10(3)/microgram DNA (pHZ132).