【目的】在青蒿植物的内生链霉菌W75中检测到一个大的环型质粒pCQ4。克隆、测序、分析和功能研究pCQ4。【方法】Southern杂交确定质粒的酶切图谱,利用接合转移和同源重组方法将质粒克隆到了大肠杆菌BAC衍生的载体上,并进行鸟枪测序和分析。【结果】获得了全长为84833 bp的pCQ4序列,预测编码129个基因,其中成簇的40个基因与其它噬菌体的基因同源。实验证明含有质粒pCQ4的菌株能够低频率释放噬菌体ФCQ4,并可以侵染消除了pCQ4的W75X孢子和形成噬菌斑。在透射电镜下观察到噬菌体颗粒,脉冲场凝胶电泳显示ФCQ4为线型DNA。pCQ4与已发表的链霉菌质粒-噬菌体pZL12比对,编码噬菌体的主要结构蛋白的基因是相似的。【结论】链霉菌大的质粒pCQ4也可以转化为噬菌体ФCQ4,其噬菌体相关的基因簇可能是可转移的单元。
[ Objective] Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4. [ Methods] Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used. [Results] The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (~CQ4) was observed by transmission electron microscopy. Linearq)CQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4. [ Conclusion] Streptomyces plasmid pCQ4 could be transformed into lytic phageФCQ4, and the phage segment on pCQ4 might be a mobile unit.