Cloning And Functional Analysis Of Transcription Factor CmMYB59 Gene

Chrysanthemum(Chrysanthemum morifolium Ramat.)is among the top ten traditional flowers in China,and occupies high ornamental and economical values for its bright color and rich flower-patterns.The MYB family of proteins is large and functionally diverse and represented in plants.In this study,we cloned CmMYB59 gene to analyze its expression profiles,transcription activities and subcellular localization.With the transgenic method,we investigated its role of root growth regulation and obtained a new germplasm of chrysanthemum,expected to lay the foundation for revealing roles of molecular mechanisms involved in root growth.The main conclusion are as follows:1.A novel MYB and its alternative splicing transcript were isolated from Chrysanthemum morifolium 'Jinba'via RT-PCR and RACE method.The full length of this gene was 904 bp,containing an open reading frame of 768 bp,encoding 255 amino acids,with a molecular weight of 61.99 kD.The multiple alignment of the protein sequences showed the protein had a typical R2R3-myb domain and was most closed to AtMYB59.Thus,the gene was named CmMYB59,Phylogenetic analysis found that CmMYB59 has a close relationship with SmMYB and ZmMYB59,respectively from Salvia miltiorrhiza and Zea mays.2.We investigated the expression level of CmMYB59 in different tissues,during the stage of flower bud differentiation and under different abiotic stresses including heat,cold,NaCl,ABA treatment via quantitative real-time PCR.RT-PCR showed the CmMYB59 expression the highest in roots,higher in stems and leaves,the lowest in shoot tip and foral organ.The CmMYB59 expression level in leaves showed a trend of rising and falling with a peak at the stage of floret primordium formation during flower bud differentiation.CmMYB59 was up-regulated in response to cold stress,and for heat,ABA.NaCl,ABA treatment,it appeared a tend which was decreased in the first 2 h,but rose quickly after then.The different expression patterns under abiotic stresses revealed that CmMYB59 was involved in stress response via different regulating mechanisms.GFP expression vector pMDC43-GFP-CmMYB59 was constructed and transformed into the onion epidermal cells.The subcellular localization indicated that the protein was located in nucleus.We constructed the vector pDEST-pGBKT7-CmMYB59 and transformed it into the yeast strains Y2H.The yeast one-hybrid assay showed that the CmMYB59 protein had a transcriptional activation function.3.We constructed the over-expression vector pMDC43-CmMYB59 and transformed it to Arabidopsis and chrysanthemum 'Jinba' by Agrobacterium-mediated genetic transformation.The positive transformants were selected by hygromycin resistance screening and qRT-PCR.The over-expressing transgenic lines Z2,Z6 of Chrysanthemum and L1,L3 of Arabidopsis were identified.We observed flowering time of the transgenic strains and wild type plants,to find that there was no significant difference between them,indicating that CmMYB59 had no direct effect on regulating flowering time.Root length measurement suggested that transgenic Arabidopsis over-expressing CmMYB59 exhibited shorter roots compared with wild-type plants.Furthermore,we investigated the expression level of some genes involved cell cycle,such as CYCB1;1,NAM-LIKE gene and ARR16,found that CmMYB59 could up-regulate CYCB1;1,NAM-LIKE gene,but had no efffect on ARR16.These results suggested that CmMYB59 could regulate plant root growth via cell cycle regulation.