Change In Content Of Associated Endogenous Hormones During Flowering Stage Of Chrysanthemum Morifolium Cv Hangbaiju And An Analysis And Identification On Their CmPEBP Family Genes

Chrysanthemum morifolium Ramat cv Hangbaiju is one of the famous traditional Chinese medicinal materials "Zhejiangbawei".Its flower is rich in nutrients and pharmacological ingredients.In this study,two elite cultivars of Hangbaiju,i.e.‘Zaoxiaoyangju’ and ‘Wanxiaoyangju’,were used to determine the dynamic changes in content of five endogenous hormones and the expression of their related genes from vegetative growth stage to full flowering stage.The changes,transport and effects of endogenous hormones in apical,stem and leaf parts during flower bud differentiation and flower organ development were analyzed.Furthermore,nine members of CmPEBP gene family closely related to flowering were cloned and identified,on which bioinformatics analysis and tissue specificity detection were carried out.At the same time,the expression patterns of seven members of CmPEBP gene family(Cm FT1,Cm FT2,Cm FT3,CmMFT1,CmTFL1,CmTFL2 and CmTFL3)under temperature stress,light treatment and dark treatment were detected.The main research contents and results are as follows:(1)The dynamic changes in content of five endogenous hormones in Hangbaiju during flowering were analyzed by HPLC,and the expression of associated genes of five endogenous hormone in Hangbaiju was detected by qRT-PCR.It was found that there were great differences in the expression levels of five endogenous hormones and five related genes in different cultivars and different developmental stages of Hangbaiju.During flower bud differentiation,the content of gibberellin(GA)in the tip,stem and leaf of cultivar ‘Wanxiaoyangju’ was always higher than that of Zaoxiaoyangju,and showed an upward,unchanged and downward trend in the tip,stem segment and leaves of the two varieties of Hangbaiju.The expression of Cm GA20 ox was up-regulated during this period.Therefore,we speculated there was a phenomenon that GA was synthesized and enriched to the tip of leaves during flower bud differentiation,the content of abscisic acid(ABA)in the tip of late daisy increased sharply during flower bud differentiation,and there was a significant difference in ABA content between the two varieties during flower bud differentiation and early flower organ development.Auxin(IAA)fluctuated at a high level among the three parts.It was found that the content of IAA at the top of the undifferentiated stage increased sharply and decreased during the development of flower organs,while the content of IAA in the stem segment changed in the opposite direction,and there was no significant change in the expression level of YUC in the stem segment,so we speculated that there may be a process of downward transport of IAA after the completion of flower bud differentiation.Thus it can be seen that GA,ABA and IAA play a unique role in the flowering process of Hangzhou white chrysanthemum.Appropriate concentrations of GA,ABA and IAA can promote flower bud differentiation and flower organ development of chrysanthemum,and high concentration of ABA can make plasmodesmata close and hinder IAA output and GA input,which may be the most important factor to delay the flowering of Wanxiaoyangju.(2)Based on the known genes of 14 plant species,9 members of CmPEBP gene family were identified from Hangbaiju transcriptome database,on which phylogenetic analysis and motif joint analysis were carried out by means of bioinformatics.It was named according to the genetic distance between CmPEBP protein sequence and Arabidopsis thaliana sequence.The PEBP gene family of Hangbaiju consists of 3 Cm FT,2 CmMFT and 4 CmTFL genes,which belong to 3groups and 5 class groups respectively.The average length of the protein sequence is173 AA.The predicted molecular weight of the protein is 18.45-20.98 k Da.The theoretical isoelectric point(p I)ranges from 4.71(CmMFT1)to 9.54(CmTFL2).The prediction of subcellular localization is mostly located in the cytoplasm and nucleus.qRT-PCR analysis of tissue-specific expression showed that the expression pattern of Gm TFL3 was similar to that of soybean Gm TFL1,and the expression was higher in young roots and stems.The expression of CmTFL4 was the highest in leaf tissue,petiole tissue and apical tissue during vegetative growth period,while the expression level of CmMFT1 was the lowest in root tissue during vegetative growth stage and flowering stage.(3)The expression patterns of 7 CmPEBP genes in response to exogenous hormone stress under light treatment and dark treatment were compared and analyzed by qRT-PCR technique.The results showed that seven different exogenous hormones had different effects on the expression of CmPEBP: IAA can inhibit the gene expression of CmTFL1;BA can promote the gene expression of CmTFL1 and CmTFL3;ABA can promote the gene expression of Cm FT2,Cm FT3 and CmTFL3;ET can promote the gene expression of Cm FT2,Cm FT3,CmTFL2 and CmTFL3;SA can inhibit the gene expression of CmTFL3;Me JA can promote the expression of Cm FT2,Cm FT3,CmTFL1 and CmTFL2.Me SA can inhibit the gene expression of CmTFL3 and promote the gene expression of CmTFL2.However,the expression of CmMFT1 was promoted by 7 hormones in the two treatment groups.Temperature stress also affected the expression of CmPEBP gene: low temperature(4℃)inhibited the expression of Cm FT1,Cm FT2 and Cm FT3,and promoted the expression of CmMFT1 and CmTFL1,CmTFL2 and CmTFL3 in different degrees,while high temperature(38 ℃)promoted the expression of four CmPEBP genes(Cm FT2,CmMFT1,CmTFL1,CmTFL2)in different degrees.Among them,the expression of CmMFT1 gene was up-regulated the most.The present work revealed the effect of endogenous hormones on the flowering process of Hangbaiju and expounded the expression characteristics of associated CmPEBP gene family in Hangbaiju,which provided references for the deep investigation and development flowering mechanism of Hangbaiju.