Harpin_(Xoo)诱发烟草过敏反应早期H_2O_2变化及有关基因表达

Harpin_(Xoo) induced hypersensitive cell death: H_2O_2 accumulation and related gene expression

HarpinXoo是水稻黄单胞细菌（Xanthomonas oryzae pv．oryzae）产生的蛋白类激发子，用harpinXoo注射烟草最早在处理后20h出现肉眼可见的细胞死亡（macro-HR），分别经埃文斯蓝（Evans blue）和台盼兰（Trypan blue）染色观察，发现诱导后4-8h即开始有少量细胞死亡，表明在macro-HR发生前烟草细胞已陆续发生微细胞死亡（micro-HR）；对二氨基联苯胺（diamino benzidine，DAB）染色显示，在烟草细胞死亡早期，伴有H2O2的明显积累；定量RT—PCR法对H2O2代谢相关基因的表达进行了分析，发现harpinXoo诱导后1h编码NADPH氧化酶的基因rboh即开始表达，3～7h表达较强，10h开始减弱，表达趋势与H2O2积累趋势一致；编码交替氧化酶的基因aoxl表达较迟，sodA基因则在诱导后表达减弱。这些基因的不同表达模式说明，它们在harpinXoo诱导的H2O2代谢过程中可能具有不同的作用。

To characterize molecular and biochemical mechanisms of the hypersensitive reaction （HR） in plants, tobacco plants （Nicotiana tobacum cv. Xanthi n. c. ） were treated with the proteinaceous HR elicitor harpinXoo from pathogenic bacteria Xanthomonas oryzae pv. oryzae. Tobacco leaves infiltrated with 40μg/mL harpinXoo developed lesions reminiscent of the hypersensitive response at the 20th hour after being treated. Using Evans blue and Trypan blue staining, we investigated the kinetics of dead cells in tobacco leaves induced by harpinXoo. The results indicated that micro-HR began to occur at the 4th hour after treatment. Since H2O2 was suggested to be a signal in cell death ,we analyzed the generation of H2O2 using DAB staining. H2O2 accumulated rapidly and obviously following harpinXoo treatment. The results of quantitative RT-PCR showed that the time course of harpinXoo induced H2O2 accumulation is similar to that of the expression of the gene rboh. Expression of aoxl is a relatively late event, but the expression of sodA is decreased after induction by harpinXoo.