摘要
利用麦迪霉素产生菌中分离的质粒pSMY1(10.8kb),将pIJ30的硫链丝菌素抗性基因(tsr)克隆到pSMYJ上,获得了具有硫链丝菌素抗性选择标记质粒pSJ10。通过DNA体外缺失,片段播入、λ-COS片段的插入及与大肠杆菌质粒的重组等基因技术,获得了一系列pSMY1衍生质粒,包括双标记质粒pSM3,大肠杆菌/链霉菌穿梭质粒pBMJ2和穿梭粘粒pNMJ1。通过对这些质粒的分析,确定了质粒pSMY1的复制必需区为3.06kd的EcoRI-SphI片段。质粒pSJ10、pSM3、pNMJ1具有可选择标记,多个单一确切的可克降位点,有一定范围的宿主并能在链霉菌中稳定存在等特点,故可作为链霉菌基因克隆的载体。其中pNMJ1(11.15kb)是大肠杆菌/链霉菌穿梭粘粒,能有效地运载28—38kb的外源DNA片段,并能在体外包装λ-噬菌体外亮,转导大肠杆菌。利用载体pNMJ1,通过λ-噬菌体蛋白体外包装,在大肠杆菌中建立了螺旋霉素产生菌的基因文库,其基因覆盖率可达90%。重组DNA分子可通过转化转移到链霉菌中。
The BamHI-fragment, containing the thiostrcpton-resistance gene obtained from pIJ30, was inserted into the plasmid pSMY1 (10.8kb) from Streptomyces mycarofaciens 1748 to obtain a plasmid pSJ10. A series of derivative plasmids from pSJ10 consisting both tsr gene and mel gene pSM3, shuttle plasmid pBMJ2 have been constructed, pBMJ2 was shown to replicate and express their resistance markers in both E. coli and Streptomyces. A E. coli/Streptomyces bifunctional cosmid vector pNMJ1 (11.15kb) which can accommodate up to 28—38kb of foreign DNA, has been constructed. By analysis of these plasmids, the essential regions of pSMY1 was determined to be in 3.06kb EcoR Ⅰ-Sph Ⅰ fragment. pNMJ1 showed a wide host range and was stably maintained in several Streptomyces species.
A genomic DNA library from spiramycin producing strain S. spiramyceticus N.sp.Yan et Yu U-1941 has been established in E. coli using pNMJ1 in vitro packaging. This rceombinant DNA can be transferred into Streptomyces.
出处
《生物工程学报》
CAS
CSCD
北大核心
1989年第4期270-278,共9页
Chinese Journal of Biotechnology
关键词
质粒
基因载体
链霉载体
链霉菌
Plasmid, cosmid, streptomyces
gcnomie library, antibiotics
