Molecular Mechanism Of CmMYB121 In Regulating Tolerance Of Chrysanthemum To Salt

Chrysanthemum(Chrysanthemum morifolium)originated in China,and it is an important ornamental flower in the world.It has high ornamental value,as well as tea,medicinal and edible value.In addition,chrysanthemum has a profound cultural heritage in China and it is widely loved by people.During the production of chrysanthemum,it is subjected to many stresses,of which soil salinization is one of serious abiotic stresses.Salt can significantly inhibit the growth and flowering of chrysanthemum,cause the plant yellowing,wilting and death.Therefore,it is of great significance to excavate the genes regulating salt tolerance in chrysanthemum and explore its molecular mechanism,which will be beneficial to the development of salt tolerant chrysanthemum germplasm.Based on previous salt stress transcriptome data,a MYB transcription factor was screened,which responded to salt stress significantly,it differentially expressed in CmHSFA4 overexpressing chrysanthemum compared with WT,therefore,it was expected as a vital gene in regulating tolerance of chrysanthemum to salt.The MYB gene was cloned and overexpressed in chrysanthemum for function identification,and its transcriptional regulation by CmHSFA4was verified.The main contents and conclusions are as follows:1.The MYB gene isolated from chrysanthemum comprised a 705 bp open reading frame(ORF),encoding 234 amino acid residues,with two conservative MYB-DNA binding domain structure,which is belonged to the R2R3-MYB family.Phylogenetic analysis confirmed that it is closely relate to the AtMYB121.So it was designated as CmMYB121.Fluorescence quantitative qPCR analysis showed that CmMYB121 expressed in different tissues of chrysanthemum,the highest abundance of the transcript presents is in the root,and it was rapidly induced by salt stress.Yeast two-hybrid suggested that it was of transactivation activities.Subcellular localization analysis showed that it was localized to the nucleus.2.In order to further study the role of CmMYB121 in the regulation of salt tolerance in chrysanthemum,The over-expression(OE)transgenic lines were obtained by transgenic transformation of wild-type‘Jinba’.OE-14 and OE-22 lines accumulated higher Na~+contents compared with wild type(WT)plant under 200 mM NaCl treat.After 14 d salt stress and 7d recovery the survival rates of OE-14 and OE-22 were 42.38%and 46.67%respectively,which is lower than that of 70.00%of WT.The expression levels of CmSOS1 in OE-14 and OE-22 are lower compared with the WT.The sensitivity of CmMYB121 transgenic lines to salt was validated through salt treatment of detached leaves,the damaged blade area percentages of OE-14 and OE-22 lines were 43.90%and 53.67%respectively,which were significantly higher than that of 19.42%of WT after salt treatment.In conclusion,CmMYB121 negatively regulates the salt tolerance of chrysanthemum.CmMYB121 may affect Na~+transport by inhibiting expression of CmSOS1,consequently negatively regulate tolerance of chrysanthemum to salt.3.In order to explore the upstream regulatory factors of CmMYB121,the promoter sequence(1,058 bp)of CmMYB121 was cloned,which contains HSE,ABRE and MYB binding motifs.CmHSFA4 protein could bind to the promoter region of CmMYB121 by yeast one-hybrid assay.EMSA assay again demonstrated that CmHSFA4 protein can specifically bind to the HSE motifs on the CmMYB121 promoter.RT-qPCR confirmed that CmHSFA4down-regulated its expression.