Study Of The Molecular Mechanism Of CmBES1 In Chrysanthemum Flowering

Chrysanthemum(C.morifolium)is an important ornamental flower.Its agronomic characteristics and ornamental value are very high,and it is favored by consumers.Flowering is one of the most important agronomic traits of ornamental flowers.The flowering period is mainly from late October to early December,and the flowering period is short and concentrated.Therefore,the regulation of the flowering time can effectively improve the commodity value of chrysanthemum to meet the market demand.BES1 protein regulates a variety of biological processes in Arabidopsis thaliana,but has not been reported in chrysanthemum.In this study,a BES1 family gene CmBES1 was cloned from chrysanthemum ’Jinba’,and the following results were obtained:1.CmBES1 was cloned from chrysanthemum ’Jinba’,encoding 306 amino acids,and containing the conserved structural domain of BES1-N at the N terminal,which has high homology with AtBES1 in Arabidopsis.The results of subcellular localization of tobacco showed that CmBES1 was located in the nucleus.Tissue specificity analysis shows that in the period of vegetative growth,CmBES1 expression in leaf is higher,followed by the stem and apex,during reproductive growth,the transcriptional level of CmBES1 was high in ray floret pistils and disc floret pistils,medium in ray floret petals,disc floret petals and roots,but low in stems,leaves and disc floret stamens.Transactivation assay in yeast and protoplast of Arabidopsis showed that CmBES1 had no transactivation activity.2.In order to verify the function of CmBES1 in chrysanthemum,an overexpression vector(pMDC43-CmBES1)was constructed,and transgenic chrysanthemum was obtained by Agrobacterium-mediated transformation.The flowering phenotype of CmBES1 overexpressing lines was observed,and it was found that the flowering time of pMDC43-CmBES1 transgenic chrysanthemum was 2 days earlier than wild-type.The third mature leaf and apex of transgenic chrysanthemum and wild type in the LD condition was collected for RNA-Seq,and found that the expression level of some flowering regulation related transcription factors CmFTL3,CmCOL,CmGA20ox,CmGA3ox2,SVP,CsLHY were changed,some genes related to the regulation of nitrogen absorption,including CmNRRa,NLP were differentially expressed.In addition,an AP2 family transcription factor AP2 was down-regulated,and the gene was cloned,with an open reading frame(ORF)of 1404 bp,encoding a total of 467 amino acids,containing two conserved AP2 domains.The results of the subcellular localization of tobacco showed that the cells were located in the nucleus,and the expression level was the highest in the disc florets.Transactivation assay in yeast and protoplast of Arabidopsis showed that CmAP2 had no transactivation activity.After genetic transformation of chrysanthemum,the phenotype of flowering period was observed,and it was found that the overexpressing lines delayed flowering,and there was no significant difference between the artificial miRNA(amiR)transgenic plant and the wild type.3.It was showed that the degree of outermost ray floret fusion of transgenic plants increased significantly at flowering stage.Further statistical results showed that the wild-type chrysanthemum outermost ray floret was mainly flat petals,accounting for 77.0%,while the spoon-shaped accounted for 23.0%,without tubular.In transgenic plants,most of the outermost ray floret were tubular(60.1%～80.0%),while the proportion of spoon-shaped petals was 20.0%～39.9%,without flat petals.In addition,the proportion of disc florets in each inflorescence of transgenic chrysanthemum also changed significantly.There was no significant difference in the total number of florets in the capitulum between WT and overexpressing lines;however,in the transgenic lines,the number of disc florets was significantly down-regulated compared to WT,and the ray florets significantly upregulated.To gain a better understanding of how CmBES1 affects the organ development.we conducted RNA-seq-based transcript profiling to identify potential genes and pathways involved in organ fusion.Analysis of differentially expressed genes screened by transcriptome for flower development showed that auxin pathway transcription factor,flower development-related MADS-box transcription factor,and boundary regulation-related genes were all differentially expressed,among which the key gene CUC2 for boundary regulation was down-regulated in transgenic lines.In order to verify whether CmBES1 directly regulates the expression of CUC2,ChIP-qPCR and LUC assay were conducted,the results show that CmBES1 can directly bind to the CUC2 promoter and inhibit its expression,it is speculated that BES1 regulation of the ray floret corolla boundary is mainly through binding to the CUC2 promoter directly to inhibit its transcription.