萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析(英文)

Radish Phospholipid Hydroperoxide GlutathionePeroxidase Gene Structure and UpstreamRegulatory Sequence Analysis

磷脂氢谷胱甘肽过氧化物酶(PHGPx)是谷胱甘肽过氧化物酶(GPx)家族的重要一员,是目前已知能直接保护生物膜免受过氧化损伤的唯一酶类.此前的研究表明,萝卜磷脂氢谷胱甘肽过氧化物酶基因(RsPHGPx)编码一个有生理功能的过氧化物酶,并且RsPHGPx基因的表达可能受发育和环境胁迫信号的复杂调控.要深入了解该基因的表达调控机制首先必须阐明RsPHGPx基因的结构及其上游调控序列.DNA印迹表明萝卜RsPHGPx基因以单拷贝的形式存在于基因组中.以基因组DNA为模板,通过常规PCR与染色体步行相结合的方法克隆到了一段3.3kb长的RsPHGPx基因组序列.分析发现,该基因由7个外显子和6个内含子组成,所有内含子的剪切位点均符合真核生物GT-AG规则.另外还发现该基因的上游基因是生物素合成酶基因;位于RsPHGPx基因上游的调控序列只有不足300bp.这些结构特征与拟南芥AtGPX3基因极其相似.顺式作用元件的数据库搜索发现RsPHGPx基因的上游调控序列含有多个响应激素(如E-Box和W-Box)、胁迫(如转录因子MYB和MYC的结合位点)和光(如BoxⅡ和Ⅰ-Box)信号的元件.RNA印迹分析表明RsPHGPx基因的表达受到脱落酸(ABA)和连续光照(在黄化苗中)处理的负调控,受到冷胁迫(4℃)的正调控,这暗示了预测的顺式作用元件的调控作用.然而,除草剂paraquat对该基因表达的正调控作用,暗示了某些与氧化胁迫相关的未知元件的存在.这些结果进一步印证了RsPHGPx基因的表达受发育和环境胁迫信号复杂调控的推测.这是迄今为止首个关于植物PHGPx基因结构和上游调控序列的系统报道,为今后全面认识植物PHGPx基因的表达调控机制奠定了必要基础.

A novel radish RsPHGPx cDNA, which encodes a functional phospholipid hydroperoxide glutathione peroxidase (PHGPx) protein, was identified in the previous work. In the study genomic organization and the upstream regulatory sequence analysis of this gene was presented. Southern blot analysis showed that RsPHGPx gene existed in radish genome in manner of single copy. Moreover, a 3.3 kb genomic DNA fragment of RsPHGPx gene was isolated by combination of common PCR and genome-walking method. Sequence analysis on this genomic fragment demonstrated that RsPHGPx gene consists of seven exons separated by six introns, and suggested that a short 5'-flanking sequence immediately before the exon I should be the putative RsPHGPx promoter region, which is proceeded by the upstream neighboring biotin synthase gene. Cis-acting elements search showed that the putative promoter contains elements responsive to hormones (eg. E-Box and W-Box), abiotic stresses (eg. MYB and MYC binding sites), and light (Box H and I-Box), etc. Northern blot analysis indicated that the expression of RsPHGPx was subjected to up-regulation of chilling and down-regulation of ABA and successive illumination (in etiolated seedlings), implying the regulatory roles of some predicted elements. However the up-regulation effect of herbicide paraquat, which can induce oxidative stress, suggested the presence of some unknown elements in the promoter region. This is the first report on gene structure and upstream regulatory sequence analysis in reported plant PHGPx genes, which will be a prerequisite to understand regulatory mechanism of PHGPx gene expression in plants.