一个大豆ERF转录因子的克隆与表达分析

Cloning and expression analysis of an ERF transcription factor in soybean

利用RT-PCR方法从聚乙二醇(PEG)处理的大豆根部组织中克隆了1个大豆乙烯响应因子(ERF)基因,由于其位于大豆基因组第7染色体上,故命名为GmERF7,基因座为Glyma07g02930。序列分析结果表明:该基因含有1个237 bp长的内含子,开放阅读框(ORF)长585 bp,编码194个氨基酸,蛋白质分子量为2.199×104,理论等电点为7.06;通过氨基酸序列比对发现,该序列与其他物种ERF类蛋白质氨基酸序列有很高的相似性;聚类分析结果表明,GmERF7与苜蓿和蓖麻遗传距离最近,与拟南芥和葡萄遗传距离相对较远;半定量RT-PCR分析结果表明该基因主要在大豆叶和豆荚中表达,而且在外源20%PEG处理不同时间后,该基因的表达在根部组织和叶片组织都受到不同程度的诱导,暗示该基因可能对提高植物耐旱性具有一定作用;利用洋葱表皮细胞的亚细胞定位分析结果表明,该蛋白质位于细胞核内,当去除N端信号肽之后,该蛋白质分布于细胞膜部位,这表明该蛋白质在植物体内通过充当转录因子的作用来调节下游基因的表达;适时定量-PCR(Real-time PCR)分析结果证实,该基因在外源ABA处理后呈现先下调后上调又下降的趋势,表明该基因受ABA的诱导,可能参与了ABA依赖的植物抗逆信号转导途径。

Total RNA was isolated from soybean root tissues treated with 20% PEG-6000 solution and reversely transcribed into cDNA using RT-PCR method.An ethylene responsive factor（ERF） gene（designated as GmERF7 because of being located on the seventh chromosome,locus name： Glyma07g02930） was cloned.The sequence analysis indicates that GmERF7 contains an intron of 237 bp and an ORF（open reading frame） of 585 bp.It encodes a protein（molecular weight： 21.99×103） of 194 amino acids with theoretical pI of 7.06.Sequence analysis of amino acids demonstrated its high similarity with those from other plant species.The phylogenetic analysis depicted it＇s nearer genetic distance with Medicago truncatula and Ricinus communis and farther with Vitis vinifera and Arabidopsis thaliana.Semi-quantitative RT-PCR showed that this gene mainly expressed in leaf and pod tissues of soybean.The expression of GmERF7 in root and leaf could be induced after 6 h of 20% PEG treatment indicating its probable function during the drought stress.The subcellular localization using onion epidermal cells showed that GmERF7 protein was mainly localized at the nucleus and cell membrane after N-terminus signal peptide,and thus indicated that GmERF7 was a regulator for downstream genes.Real-time RT-PCR analysis indicated the down-regulation of gene at 10 min,then up-regulation at 30 min followed by down-regulation up to 2 h of exogenous application of ABA,implying that the gene maybe participate in transduction pathway of ABA-dependent signal for plant tolerance to adverse environment.