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    孙丽萍, 沈琼, 叶江, 吴海珍, 张惠展. 红色糖多孢菌红霉素抗性基因的克隆与鉴定[J]. 华东理工大学学报(自然科学版), 2002, (1): 20-23.
    引用本文: 孙丽萍, 沈琼, 叶江, 吴海珍, 张惠展. 红色糖多孢菌红霉素抗性基因的克隆与鉴定[J]. 华东理工大学学报(自然科学版), 2002, (1): 20-23.
    Cloning and Identification of Antibiotic Resistance Gene of Saccharopolyspora Erythraea[J]. Journal of East China University of Science and Technology, 2002, (1): 20-23.
    Citation: Cloning and Identification of Antibiotic Resistance Gene of Saccharopolyspora Erythraea[J]. Journal of East China University of Science and Technology, 2002, (1): 20-23.

    红色糖多孢菌红霉素抗性基因的克隆与鉴定

    Cloning and Identification of Antibiotic Resistance Gene of Saccharopolyspora Erythraea

    • 摘要: 将红色糖多孢菌(Saccharopolyspora erythraea)的染色体DNA用PsTi酶切后与载体PUWL201连接,转化变沿青链霉菌(S.lividans)TK23的原生质体。采用双重抗性筛选得到了9个转化子。分别抽提其中的质粒并再次转化原生质体,在含红霉素的平板上各筛选约200个转化子,其中3个在抗性板上生长良好,分别命名为PLP42、PLP1b和PLP44。对其中的PLP42酶切分析表明,其含有约3.0Kb的红色糖多孢菌(Saccharopolyspora erythraea)染色体DNA片段,它的载体部分发生了缺失。以PUC18为载体,将PLP42的1.7kb-KpnI片断克隆、测序、经与Genebank的ermE基因顺序比较,证实巳克隆了Saccharopolyspora erythraea的抗性基因。

       

      Abstract: Saccharopolyspora erythraea chromosomal DNA digested extensively with PstI has been cloned in shuttle vector pUWL201, which is transformed into protoplast of Streptomyces lividans TK23. Nine transformants are obtained by double antibiotic resistant screening. The nine plasmids are extracted and re transformed, then about 200 transformants are screened on plate containing erythromycin, respectively. It is found that three transformants grow well on resistant plate, designated pLP42, pLP1b and pLP44, respectively. With the electrophresis analysis of restriction fragments of pLP42, our experiment shows that pLP42 contains about 3.0kb foreign fragment from S.erythraea chromosomal DNA, and a section of vector pUWL201 has been deleted. A 1.7kb KpnI fragment from pLP42 has been cloned and sequenced. Compared with the ermE sequence in genebank, the cloned S.erythraea chromosome fragment firmly contains the antibiotic resistance gene.

       

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