十字花科黑腐病菌中一个假定双组份系统的表达和磷酸传递研究.doc

十字花科黑腐病菌双组份系统的摘要十字花科黑腐病菌Xanthomonas campestris pv. campestris，Xcc)8004，研究微生物与相互作用的模式菌基因106个ET-30a(+)/ pQE-30 Xa表达载体，在E.coli BL21(DE3)/M15[pREP4]中表达，同时对HrpG的可溶性表达进行优化，通过调节诱导温度、IPIG浓度和诱导时间最终确定HrpG的最优表达条件为20℃、IPTG浓度0.8 mM、诱导时间4 h，此时HrpG在破胞上清液的可溶性表达状况最好。纯化重组蛋白进行in vitro磷酸化实验，结果表明XC3670具有自激酶活性，能够接受[γ-32P]ATP的磷酸基团，去除HAMP和PAS结构域后(XC3670C)，对XC3670的活性不产生影响。分别检测了肉桂酸、FAD及黑暗条件等对磷酸传递的影响，结果没有检测到磷酸基团从XC3670/ XC3670C传递至XC3669/ XC3669N；使用cross-talk方法验证XC3670/ XC3670C和其他12个应答调节蛋白的磷酸传递，结果表明没有检测到磷酸传递。分析可能是由于XC3670由于在进化过程中的插入突变或其本身的严谨性以及其他原因导致磷酸传递未发生。 关键词：十字花科黑腐病菌 双组份系统in Xanthomonas campestris pv. campestris ABSTRACT Two-component system(TCS) plays important role in signal transduction pathways of microorganisms. After perceiving environment stimulus , signal is transducted to cytoplasm by phosphotransfer mechanism, resulting in response regulation accordingly. Xanthomonas campestris pv.campestris (Xcc) 8004 is one of the model bacteria in studying the interaction between microbes and plants, its genome has been sequenced, including 106 genes annotated two-component system proteins. XC3670-XC3669 which is annotated histidine kinase(HK) and response regulator(RR) respectively, is a putative two- component system. XC3670 is located downstream of XC3669, with only 4 nucleotides between them. Homology analysis shows that XC3670-XC3669 unit is conserved, and their homologies are found in many sequenced genomes. DNA sequence analysis showed that the G2 box was isolated from kinase core of ancestor XC3670 by a insertion sequence which encodes two IS1404 transferases which are over-lapped genes. In this experiment, XC3670/XC3670C, XC3669/XC3669N andhrpG are constructed pET-30a(+)/pQE-30 Xa expression vector respectively, and are expressed in E. coli BL21(DE3)/ M15[pREP4]. In order to optimize the expression ofhrpG, tempreture, IPTG concentration and time for induction is optimized, respectively. Expression of solublehrpG in supernatant of cell disrution is best while combination E.coli M15[pR