天蓝色链霉菌质粒SCP2的复制及调控的分子机理研究

Streptomyces are high GC genome content Gram-positive bacteria.Over 6000 antibiotics are produced by Streptomyces species. SCP2 is the first identified circular plasmid in model organism--Streptomyces coelicolor A3(2). SCP2 is stably inherited, and has the ability to accept large cloned fragments of exogenous DNA, which makes SCP2 the only widely used autonomously replicating vector for expressing heterologous gene clusters. However, the mechanism of SCP2 replication and regulation are not known clearly. Our group have detected the specific binding between replication protein and the non-coding sequence,and the multimerization of replication and regulation proteins in vitro. This project will characterize the mechanism of SCP2 replication and regulation, including determination of the minial functional replication region, the transcription start of replication genes, the interaction between the replication and regulation proteins and their impact on the copy number and stability of SCP2. Base on these results above, a model of replication and regulation will be established.

链霉菌是高GC含量革兰氏阳性细菌，已报道链霉菌的不同种产生约6000种抗生素和生理活性物质。SCP2是链霉菌属模式生物天蓝色链霉菌中第一个发现的环型质粒。SCP2的遗传稳定性高，宿主范围广并且具有容纳大片段DNA的能力。在链霉菌中，SCP2是目前唯一广泛应用的异源表达大片段生物合成基因簇的自主复制型载体。但是对于SCP2的复制和调控的分子机理的研究尚处于初级阶段。本实验室通过体外生化试验鉴定了SCP2复制蛋白特异的DNA结合区域，并发现了其复制蛋白和调控蛋白存在自身多聚化现象。本项目拟在遗传和生化水平上精细分析SCP2的复制和调控机制，包括鉴定最小复制功能区，复制基因的转录起始位点，复制和调控蛋白的相互作用关系及其和DNA序列的精确结合位点，调控蛋白对质粒复制和拷贝数的影响和可能机制等，并在此基础上建立复制和调控的模型。