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为研究从玫瑰黄链霉菌Men-myco-93-63中克隆到的,与天蓝色链霉菌M 145中的一个重要负调控基因nsd A基因同源的nsdA_(mgh)基因的功能,本文构建了nsd A_(mgh)基因破坏型重组质粒pSRNA2500(pKC1139::1.5 kb nsdA_(mgh)::1.0 kb Km~r),转化ET12567(pUZ8002)获得接合转移供体菌ET12567(pUZ8002,pSRNA2500),通过接合转移将重组质粒导入玫瑰黄链霉菌Men-myco-93-63中。在高温和抗生素双重筛选压力下,筛选得到表型为Am~sKm~r的nsd A_(mgh)基因阻断突变株,通过PCR、Dot bloting和Southern blotting验证了突变株中的nsdA_(mgh)基因已被正确阻断。与出发菌株相比,突变株在摇瓶水平上对棉花黄萎病菌的抑制能力提高了一倍。
In order to study the function of the nsdA mgh gene cloned from Streptomyces roseoflavus Men-myco-93-63 which is homologous to the nsd A gene, an important negative regulator of Streptomyces coelicolor M 145, (pUZ8002, pSRNA2500) was obtained by transforming ET12567 (pUZ8002) with the recombinant plasmid pSRNA2500 (pKC1139 :: 1.5 kb nsdA_ (mgh) :: 1.0 kb Km ~ r) of nsd A mgh gene, Transfer The recombinant plasmid was introduced into Streptomyces roseoflavus Men-myco-93-63. The nsdA (mgh) gene mutant with the phenotype of Am ~ sKm ~ r was screened under the dual screening pressure of high temperature and antibiotic. The nsdA mgh gene was confirmed by PCR, Dot blotting and Southern blotting Has been correctly blocked. Compared with the starting strain, the mutants doubled their ability to inhibit Verticillium dahliae at shake flask level.