Nuclear Factor CmNF-YB Regultes Flowering Time And Drought Tolerance In Chrysanthemum

Chrysanthemum is an important ornamental plants in the world,and widely used as cut flowers,potted flower,courtyard greening and landscaping.Chrysanthemum is a short day plant,the production of chrysanthemum mainly depends on the facility cultivation,so water supply and light control which need energy consumption,are the limited cost factors.Understand the mechanism of drought stress response and the flowering has important practical significance.Previous research has shown that nuclear factor Y(NF-Y)B can significantly improve crop drought stress tolerance,and participate t flowering process in a variety of plants,but little is known about how NF-YB regulates drought tolerance and flowing.To anlalyze the function of CmNF-YB8,we isolated the CmNF-YB8 gene,and analysed its expression pattern.By RNAi in chrysanthemum and overexpression in Arabidopsis,we investigated the role of CmNF-YB8 gene in drought torlerance and flowering.The res?lts are shown as bellows.1)We obtained a 952 bp CmF-YB8 gene from chrysanthemum.The deduced amino acid sequence contains a NF-YA interaction domain,a NF-YC interaction domain and a DNA binding domain.By qPCR analysis,we detected the expression of CmNF-YB8 gene in almost all the tissues,with higher expression in the shoot tips and flowers,and the lower expression in the root and stem among the tissues.The expression of CmNF-YB8 was strongly suppressed by low temperature and dehydration,but not by salt stress and ABA treatment.Through the leaf protoplasts transient expression systerm,we confirmed that CmNF-YB8 localized in the nuclear.2)We obtained CmNF-YB8-overexpressed Arabidopsis and RNAi-chrysanthemum.Compared with WT plants,RNAi chrysanthemums flowered earlier with no other obvious differences.Flowering time of CmNF-YB8 overexpressed Arabidopsis was late and the inflorescences of transgenic plants were significantly longer than that of control plants.Statistically,the inflorescence was caused by the increased internode number but not internode length.3)To investigate the functional role of CmNF-YB8 gene in drought tolerance,we treated the transgenic chrysanthemum and Arabidopsis plants with drought stress.The res ? Its showed that,compared with control,CmNF-YB8-overexpressed plants decreased the tolerance to drought stresses,and RNAi plants increased the tolerance.4)It was confirmed that CmNF-YB8 influences flowering time through regulating the expression of CO and SPLs,the maker gene of photoperiod pathway and ageing pathway,respectively.We isolated 15 CmNF-YB8 up-regulated flower related genes in chrysanthemum by RNA-seq,including 4 CONSTANS family member,4 SPL family member and 7 Floral Pathway Integrators.We also obtained 17 differencial expressed gene,including 6 down-regulated miR156 family member by miRNA RNA-seq.Furthermore,electrophoretic mobility shift assay(CHIP)validated the direct binding of CmNF-YB8 protein to the miRNA156a and miRNA156c promoter in vivo,indicating that miRNA156a and miRNA156c are direct downstream genes to CmNF-YB8.5)RNA-seq showed 758 differencial-expressed genes in RNAi chrysanthemum,including 687 up-regulated genes and 71 down-regulated genes.These genes can be divided into two classes,regulatory protein and functional protein.By classifying these genes,we found that CmNF-YB8 responsed to drought stress through neither ABA-dependent nor ABA-independent pathway.CmNF-YB8 responsed to drought stress through regulating possible downstream genes related to hormone related protein,protein kinase and metabolism related proteins.Taken together,CmNF-YB8 regulates flowering time through decreasing the expression of SPLs by direct binding the promoter of miR156a and miR156c and responseds to drought stress through regulating hormone-related proteins,protein kinase and metabolism related proteins.