Cloning And Functional Analysis Of BrERF2 Gene In Chinese Cabbage

Chinese cabbage（Brassica rapa L.ssp.pekinensis）,which originated in China,is a leafy vegetable belonging to the genus Brassica in the cruciferous family.As the main edible organ,the leafy head not only has good taste and abundant nutrients,but also its shape directly influences the head quality and commercial value.Therefore,it is important to explore the molecular mechanism of leafy head formation.It has been found that BrERF2 gene is one of the differentially expressed genes in Chinese cabbage in the two periods before and after the heading process.In this study,we analyzed the function of BrERF2 using multiple approaches to explore the role of BrERF2 during leafy head formation,including BrERF2 bioinformatics analysis,ectopic overexpression and promoter analysis in Arabidopsis,and subcellular localization in Benthamiana.The main results of this study are as follows:The BrERF2 gene was cloned from Chinese cabbage“Bre”.Sequence analysis revealed that the 738 bp full-length sequence encoding 245 amino acids,with a relative molecular mass of 27.05k Da and an isoelectric point of 4.96.The protein sequence possessed a typical AP2 structural domain.The homologous genes in Arabidopsis and Kale was obtained to investigate the conserved motif and phylogenetic relationships.The DX2181G-BrERF2-p overexpression vector was constructed and transfected into Agrobacterium tumefaciens GV3101,which transformed into Arabidopsis.T₃ generation overexpressed lines were used to conduct GUS staining,which showed that GUS was most highly expressed in roots in rosette stage,followed by leaves,and less in stems and flower buds.The BrERF2 promoter is a constitutive promoter that contains a variety of phytohormone response elements,light response elements,low temperature response elements,etc.It may drive the BrERF2gene to participate in a variety of plant hormone signaling pathways,plant resistance to stress pathways,photosynthesis pathways,and other signaling pathways for plant growth and development.The pMDC32-35S-BrERF2 overexpression vector was constructed and transferred into Agrobacterium tumefaciens GV3101,which was transfected and screened to obtain stably pure transgenic plants.Overexpressing plants showed significant leaf curling,and the root development was consistent with WT at the seedling stage but more developed than WT at the fruiting stage.The expression level of BrERF2 was significantly higher in overexpressing Arabidopsis plants,and the IAA content in the leaves was also significantly higher than WT.It indicates that the BrERF2 gene may influence the IAA content and the distribution of different parts in the leaves by regulating the IAA signaling pathway,and then participate in the leaf formation process.The pCambia1380-BrERF2-GFP subcellular localization vector was constructed and transferred into Agrobacterium tumefaciens GV3101,which was infiltrated and transformed into Benthamiana.The localization results showed that the BrERF2 gene was expressed in the nucleus and the expression might be regulated by the nucleus.