十字花科黑腐病菌中一个假定双组份系统的表达和磷军酸传递研究.docVIP

PAGE IV 十字花科黑腐病菌中一个假定双组份系统的 表达与磷酸传递研究 摘 要 双组份系统是微生物重要的信号转导途径，在感知环境刺激后，通过磷酸传递的方式将信号传递至胞内，从而产生应答调控功能；十字花科黑腐病菌(Xanthomonas campestris pv. campestris，Xcc)8004，是研究微生物与植物相互作用的重要模式菌，全基因组测序已经完成，其中注释为双组分系统的基因有106个。XC3669，XC3670分别注释为应答调节蛋白和组氨酸激酶，是一个假定双组份系统。XC3670位于XC3669下游，两个ORF间隔4bp。同源性分析表明，XC3670-XC3669很保守，在许多已测序的微生物基因组中都存在其同源基因对，DNA序列分析表明原始XC3670的G2 box被一段插入序列所隔开，该插入序列编码两个IS1404转座酶，为重叠基因。本实验中，分别构建了XC3670/XC3670C、XC3669/XC3669N和HrpG的pET-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/ XC3669 关键词：十字花科黑腐病菌 双组份系统 插入序列 磷酸传递 EXPRESSION AND phosphotransfer ANALYSIS OF A PUTATIVE TWO-COMPONENT SYSTEM 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-