北里链霉菌(Streptomyces kitasatoensis 2488)启动子和柱晶白霉素抗性基因的克隆

用启动子探针质粒pIJ486作载体,变青链霉菌TK24作受体,克隆来自北里链霉菌SK2488的启动子和柱晶白霉素抗性基因,得到三个转化子其中转化子变青链霉菌TK24(pRL1)中重组质粒插入片段约为10kb,其外源启动活性和柱晶白霉素抗性较低;转化子变青链霉菌TK24(pRL2)中重组质粒插入片段较小,但具有高活性的外源启动子,并有较高的柱晶白霉素抗性;转化子变青链霉菌TK24(pRLX)中未分离得到重组质粒,但它同样具有区别于受体的对新霉素和柱晶白霉素的抗性,而且它在R_2YE平板上能抑制其它转化子生长,其发酵液经薄层层分析发现有不同于供体和受体的新斑点,发酵液经高压液相分析发现有一个吸收峰。但三个转化株发酵液均无抗菌活性,将重组质粒pRL1和pRL2再转化变青链霉菌TK24,抗性稳定。

CLONING OF PROMOTORS AND LEUCOMYCIN RESISTANT GENES FROM STREPTOMYCES KITASTOENSIS 2488

Promotor-probe plasmid pH486 was used as the vector and S. lividans TK24 as the host. The promotors and Leucomycin resistant genes from S. kitasatoensis 2488 were cloned. pH486 and the donor chromosome were digested with BamHI respectively. Then they were linked with T_4DNA ligase. After transformation and regeneration, three transformants were obtained. Two of them contained recombinant plasm-ids:S. lividans TK24(pRL1)and S. lividans TK24 (pRL2). The inserted fragment of recombinant plasmid pRL1 contained in S. lividans TK24 (pRL1) was about 10kb,and its promotion activity and Leucomycin resistance was low. The inserted fragment in recombinant plasmid pRL2 contained in S. lividans TK24(pRL2) was high.From the third transformant S. lividans TK24 (pRLX), recombinant plasmid was not isolated, but still it had Neomycin and Leubomycin resistance different from the recipient. And on the R_2YE plate, S. lividans TK24 (pRLX) could inhibit the other transformants' growing.The fermentation broth of it was found new blots according to TLC which were different from donor and recipient. Absorption peak appeared in the HPLC map of S. lividans TK24 (pRLX) fermentation broth. But all fermentation broth of the three transformants had not any antibactertal activities. The results of retransformation of recombinant plasmid pRLl and pRL2 into S.lividans TK24 were the same as above.