Functional Analysis Of Transcription Factors CmCUC2 And CmCUC3 In Chrysanthemum

CUCs(CUP-SHAPED COTYLEDON)belong to plant-specific NAC family transcription factors.These genes play an important role in the establishment of plant organ boundaries,morphogenesis,and flower development,and they are regulated by miR164.Previous studies of CUCs mainly focused on model plants Arabidopsis and Oryza sativa.Subsequently,the CUC homologs NOAPICAL MERISTEM(NAM),CUPULIFORMIS and GOBLET genes in petunia,snapdragon and tomato have also been cloned in succession,but there is no report in the chrysanthemum.In this study,Arabidopsis and chrysanthemum’Jinba’ were used as materials to analyze the expression characteristics and function of the chrysanthemum CmCUC2 and CmCUC3 genes.The results are as follows:1.The CmCUC2 gene is cloned from the chrysanthemum ’Jinba’ transcriptome with a total length of 1,014 bp,encoding 338 amino acids;the CmCUC3 gene is 1,128bp,encoding 376 amino acids.Phylogenetic analysis of amino acid sequences showed that CmCUC2 had the closest relationship with HaCUC2 in Helianthus annuus,and CmCUC3 had the closest relationship with AmNAC in Artemisia annua.Amino acid sequence alignment showed that CmCUC2/3 contained a NAC domain and had DNA binding function.It shows that CmCUC2/3 can directly bind to the promoter of the target gene and regulate the transcription of downstream genes.2.The tissue expression analysis of the chrysanthemum CmCUC2/3 genes revealed that CmCUC2 and CmCUC3 were the most expressed in the disc flower stamens,followed by leaves,ray flower pistils,disc flower petals and disc flower pistils,in the stem and ray flower petals has the lowest level of expression.CmCUC2/3 proteins are localized in the nucleus,and CmCUC2/3 have transcription activation activity.3.Through the heterologous transformation model plant Arabidopsis,CmCUC2 transgenic Arabidopsis lines were obtained,and they were observed that their cotyledon edges were serrated,true leaves were heart-shaped and had multiple apical meristems and ectopic inflorescence developing on a cauline leaf.Stem leaves and petal edges curled.Quantitative analysis of transgenic Arabidopsis found that the expression levels of genes auxin pathway-related PIN1,PIN5,PIN7,AUX1,LAX2 and IAA9 were significantly down-regulated.This indicates that the CmCUC2 gene may affect the development of leaves and flowers by regulating the expression of auxin-related genes and ultimately affecting the distribution of auxin.Observation revealed that CmCUC3 transgenic Arabidopsis flowered earlier,and it was speculated that CmCUC3 had the effect of early flowering.4.Agrobacterium-mediated leaf disc transformation of chrysanthemum ’Jinba’ to obtain transgenic lines.Observation found that during the vegetative growth period,OX-mCmCUC2 transgenic lines showed shorter plant heights,shorter intemode distances,deeper leaf cracks,and leaf edges curled phenotype.During the flowering period,the tip of the tray flower petals of the OX-mCmCUC2 transgenic line split,resulting in a change in flower shape and stamen petals;and the number of ray flowers decreased,deformed small flowers appeared,and more than 5 petals and petals appeared on the top of the disc flower that appears white color.The ray flower petals of the RNAi-CmCUC2 transgenic chrysanthemum are fused at the base.OX-CmCUC3 transgenic chrysanthemums showed increased branching and lighter leaves.In summary,CmCUC2/3 is highly expressed in chrysanthemum leaves and floral organs,CmCUC2 regulates the morphological formation of leaves and floral organ development,and CmCUC3 regulates branch development.They play an important role in the entire life cycle of chrysanthemums.