水稻白叶枯病菌ahpC基因转录单元分析和原核表达

Transcriptional Regulation Analysis and Prokaryotic Expression of ahpC Gene Putatively-encoding Alkyl Hydroperoxide Reductase in Xanthomonas oryzae pv.oryzae

从水稻白叶枯病菌（Xanthomonas oryzae pv．Oryzae，Xoo）菌株PX099“中克隆了H202降解基因ahpC，发现其编码的烷基过氧化氢酶AhpC在所测定的不同种病原细菌中的蛋白序列高度保守；采用RT—PCR方法分析了基因的转录结构特征，发现aheC基因与酶电子供体基因ahpF组成了同一个转录单元；通过对ahpCp—lacZ活性检测，发现该启动子活性显著地受转录调控因子OxyR的正调控。此外，利用表达载体pET-28a（＋）对ahpC基因进行了原核表达，经诱导后获得了可溶性的靶蛋白，可用于后续的生物学功能的分析。

The ahpC gene involved in degradation of hydrogen peroxide was cloned from the wild-type strain PXO99A of Xanthomonas oryzae pv. oryzae in this study. The gene putatively encodes the alkyl hydroperoxide reductase AhpC. The sequence of AhpC protein is highly conserved in different kinds of pathogenic bacteria tested. To find out the structural characteristics of transcription of the gene cluster oxyR-ahpF- ahpC, reverse transcription PCR （ RT-PCR ） analysis revealed that ahpC and ahpF gene, the enzyme electron donor gene are co-transcribed in the same operon and same transcriptional unit. The activity of ahpCp-lacZ was detected, suggesting the ahpC promoter activity was positively regulated by transcriptional regulator factor OxyR. In addition, construction of expression vector of ahpC using pET-28a （ ＋ ） and prokaryotic expression was performed. The soluble recombinant proteins were expressed, which could be purified and used for further biological function analysis.