油橄榄AP2/ERF转录因子鉴定及水胁迫表达分析

Identification and expression analysis of AP2/ERF transcription factor under water stress in Olea europaea

为探究油橄榄AP2/ERF基因家族对水胁迫的响应机制,该研究对受干旱及水淹胁迫的‘佛奥’和‘TYZ-1号’2个品种的根和叶进行转录组测序,并对油橄榄中AP2/ERF转录因子的蛋白理化性质、基因结构及系统进化进行分析,同时分析与水胁迫相关的AP2/ERF转录因子在2个油橄榄品种中的基因表达差异且进行RT-qPCR验证。结果表明:(1)在油橄榄中鉴定得到110个AP2/ERF基因家族成员,该110个蛋白质所含氨基酸大小为173~717 bp,均不存在信号肽,为非分泌蛋白。将油橄榄AP2/ERF与模式植物拟南芥AP2/ERF蛋白构建进化树发现,油橄榄AP2/ERF蛋白分为AP2、RAV、ERF和Solosist 4大类,其中ERF分为ERF和DREB 2个亚类,ERF包含ERF B1~ERF B66个子亚类,DREB包含DREB A1~DREB A66个子亚类,这与模式植物拟南芥AP2/ERF的分类一致,每个亚家族同时包含了油橄榄和拟南芥AP2/ERF蛋白,说明拟南芥和油橄榄的AP2/ERF家族在进化水平上有一定的相似性。(2)油橄榄AP2/ERF同一亚家族蛋白具有相同的基因结构及保守元件,结合基因表达和进化树中已知水分调控功能的基因初步推测OeAP2-75、OeAP2-97、OeAP2-101、OeAP2-23、OeAP2-13与油橄榄水分调节密切相关,OeAP2-13、OeAP2-28、OeAP2-104、OeAP2-75、OeAP2-80、OeAP2-50在2个品种中都有不同的表达量,推测可能是‘佛奥’和‘TYZ-1号’抗性不同的原因。(3)利用RT-qPCR技术检测油橄榄AP2/ERF基因在不同胁迫下的表达变化,OeAP2-101、OeAP2-28、OeAP2-42受水胁迫诱导显著上调,这与转录组分析结果一致。该研究结果为油橄榄AP2/ERF家族基因的抗逆性表达及基因功能研究奠定了基础,为油橄榄选育抗旱和耐涝砧木品种提供了方法和理论依据。

In order to explore the response mechanism of AP2/ERF gene family in the water stress of O.europaea,this study performed transcriptome sequencing on the roots and leaves of two cultivars‘Frantoio’and‘TYZ-1’that were under drought and flooding stresses.And based on the whole genome data,the protein physicochemical properties,gene structure and system evolution of AP2/ERF transcription factor in O.europaea were analyzed.At the same time,the difference in gene expression of AP2/ERF transcription factor related to water stress in the two O.europaea cultivars was analyzed by transcriptome sequencing data and verified by RT-qPCR.The results were as follows:(1)A total of 110 AP2/ERF gene family members were identified in O.europaea.The amino acid size of the 110 proteins was 173-717 bp,there was no signal peptide and it was a non-secreted protein.The phylogenetic tree was constructed between O.europaea AP2/ERF and model plant Arabidopsis AP2/ERF protein.It was found that O.europaea AP2/ERF protein was divided into four categories,AP2,RAV,ERF and Solosist.Among them,ERF was divided into two subtypes,ERF and DREB.ERF included six subtypes of ERF B1 to ERF B6,and DREB included six subtypes of DREB A1 to DREB A6,which was consistent with the classification of the model plant Arabidopsis AP2/ERF.Each subfamily contained AP2/ERF proteins of O.europaea and Arabidopsis at the same time,indicating that the AP2/ERF family of Arabidopsis and O.europaea were similar in evolution.(2)The analysis of gene structure and conserved elements found that the proteins of the same subfamily of O.europaea AP2/ERF had the same gene structure and conserved elements.Combining gene expression with genes with known water regulation functions in the evolutionary tree,it was preliminarily speculated that OeAP2-75,OeAP2-97,OeAP2-101,OeAP2-23 and OeAP2-13 were closely related to the water regulation of O.europaea,OeAP2-13,OeAP2-28,OeAP2-104,OeAP2-75,OeAP2-80 and OeAP2-50 had different expression levels in the two cultivars.It is speculated that this may be the reason for the different resistance of‘Frantoio’and‘TYZ-1’.(3)The RT-qPCR technique was used to detect the expression changes of O.europaea AP2/ERF gene under different stresses.The results showed that OeAP2-101,OeAP2-28 and OeAP2-42 were significantly up-regulated by water stress,which was consistent with the results of transcriptome analysis.The results of this study lay a foundation for the research on the stress resistance expression and gene function of the AP2/ERF family genes of O.europaea,and provides the method and theoretical basis for the selection of drought-resistant and flood-tolerant rootstock cultivars of O.europaea.