摘要
以启动子探针质粒ｐＩＪ４８６为载体，变铅青链霉菌（Ｓ．ｌｉｖｉｄａｎｓ）ＴＫ２３作受体，克隆了林可链霉菌噬菌体ＳＬ６０ＤＮＡ上的启动子活性片段。以ｐＵＣ１８为载体构建ＳＬ６０ＤＮＡ之ＫｐｎＩ片段的基因文库，从中筛选出两个片段，分别克隆于ｐＩＪ４８６中获得ｐＵＩＳ２９和ｐＵＩＳ１９８，在ＭＭ培养基上，两者的转化子对卡那霉素的抗性均仅为１０ｍｇ／Ｌ。噬菌体ＳＬ６０ＤＮＡ的ＢｇｌＩ片段直接克隆入ｐＩＪ４８６的ＢａｍＨＩ位点，获得重组质粒ｐＭＭＳ１，其转化子的卡那霉素抗性在ＭＭ培养基上可达２５０ｍｇ／Ｌ。分析表明ｐＭＭＳ１上的插入片段约为９２０ｂｐ，含有一个ＳｓｔＩ位点，四个ＭｂｏＩ位点。该片段用ＭｂｏＩ部分酶解缩小到６００ｂｐ，亚克隆入ｐＩＪ４８６中得到重组质粒ｐＭＳＢ１４，其转化子对卡那酶素的抗性水平提高到３００ｍｇ／Ｌ。ＳＤＳ－聚丙烯酰胺凝胶电泳显示，ｐＭＳＢ１４中的ｎｅｏ基因可以表达出氨基糖苷磷酸转移酶（ＡＰＨＩ蛋白）。这些结果表明ｐＭＳＢ１４中的插入片段可能具有较强的启动子活性。
Using promoterprobe plasmid pIJ486 as vector, S.lividans TK23 (tsr-,neo-) as host, promoters from actinophage φSL60 DNA were cloned and characterised. Phage φSL60 DNA KpnI fragments were cloned in E.coli plasmid pUC18 at first, then the fragments containing promoter activity were screened. Recombinant plasmids which have phage DNA fragment and plasmid pIJ486 were both digested by EcoRI/HindIII,then ligated and transformed into S.lividans TK23. Transformants harboring recombinant plasmids, pUIS29 and pUIS198, showed resistance both to thiostrepton and kanamycin when selecting on MM agar medium containing 5mg/L of kanamycin. But the maximum resistance level of these transformants are only to 10mg/L of kanamycin. Promoter activity was also screened by shotgun cloning. Phage φSL60 DNA was digested by BglII and pIJ486 by BamHI, then ligated and transformed into S. lividans TK23. Transformants harboring recombinant plasmid pMMS1 resistant to 250mg/L of kanamycin was gained while selecting on MM agar medium. The fragment inserted in pMMS1 was 920bp in size and had 1 SstI site, 4 MboI site. The 920bp fragment was partialy digested by MboI and then subcloned into pUC18. pMSB14 with a 600bp MboI fragment was then acquired. The transformant containing pMSB14 showed resistance to 300mg/L of kanamycin. The SDSPAGE showed that crude extracts from S.lividans(pMSB14) had a different lane (probably APHII) comparing to the control S.lividans(pIJ486). All of the above results indicated that the 600bp fragment might contain a strong promoter.
出处
《华东理工大学学报（自然科学版）》
CAS
CSCD
北大核心
1998年第4期415-421,共7页
Journal of East China University of Science and Technology
基金
国家自然科学基金
关键词
链霉菌
启动子
林可链霉菌
噬菌体
克隆
promoter
streptomyces lincolnensis phage φSL60
kanamycin resistance
