甘蓝型油菜谷胱甘肽过氧化物酶基因的克隆及表达分析

摘要

为了解谷胱甘肽过氧化物酶基因在油菜抗非生物逆境中的作用,采用RT-PCR技术从甘蓝型油菜(Brassica napus L.)宁油16号中克隆到谷胱甘肽过氧化物酶基因BnGPX2的cDNA全长序列644 bp,包括510 bp的开放阅读框,推测编码169个氨基酸残基,蛋白分子量为18 960,等电点为5.60.序列比对结果显示BnGPX2蛋白序列与拟南芥AtGPX2有较高的同源性(92.3％),具有植物GPX特有的3个典型结构域及Cys残基.通过常规PCR方法克隆到BnGPX2基因组序列1 782 bp,该基因由6个外显子和5个内含子组成,且所有内含子的剪切位点符合真核生物GT-AG规则.半定量RT-PCR法检测BnGPX2在油菜不同器官及非生物逆境下的表达,结果发现BnGPX2在根、茎、叶和花中表达量较高,角果中较低.在湿害和干旱胁迫下,BnGPX2表达量有所升高,但出现峰值的时间不同,而盐、高温和低温胁迫对BnGPX2表达量没有明显影响.%In order to investigate the role of glutathione peroxidase (GPX) gene in rapeseed ( Brassica napus L. ) under abiotic stress,the BnGPX2 gene cDNA was cloned from rapeseed variety Ningyoul6 by RT-PCR. It was 644 bp in full length, and its open reading frame was 510 bp in length, which encoded a 169 amino acids sequence with isoelectric point of 5.6 and molecular mass of 18 960. The deduced protein sequence was very similar(92.3% ) to the GPX2 of Arabidopsis thaliana , and had three characteristic domains of plant GPXs and three conserved Cys residues through alignment of the whole amino acid sequences of some known GPXs. The genomic DNA fragment of BnGPX2 gene was isolated by common PCR method, being 1 782 bp in full length. Sequence analysis of the genomic fragment demonstrated that the gene consisted of six exons and five introns, and all the in-trons were spliced following the consensus sequence, having GT and AG at their 5'and 3'ends, respectively. Expression analysis of the BnGPX2 in rapeseed tissues was performed by semi-quantitative RT-PCR, and different patterns were detected under different abiotic stresses. The results showed that the BnGPX2 was constitutively and u-biquitously expressed, with high levels in root, stem, leaf or flower, and low in silique. The expressions of BnGPX2 increased under drought or water logging stress. However, it did not respond to the salt, high temperature or cold stress.