Study On Regulation Mechanism Of MtDof32 On Organ Development And Branching In Medicago Truncatula

As model plant of leguminosae,Medicago truncatula has a small genome size and relative ease genetic transformation that is widely used in fundamental research of molecular biology.The research data are representative and provide genetic basis for the variety improvement of other legumes.Dof（DNA binding with one Finger）family transcription factors are a class of zinc finger proteins unique in plants.The proteins structure contains a unique Dof conserved domain,which usually composed with DNA binding domain and transcriptional regulatory domain.The amino acid sequence of transcription regulatory domain is more variable,which makes the function of Dof proteins more diverse.Studies have shown that Dof proteins play important roles in plant growth and development,involving flowering regulation and organ development.Although there have been many studies on the function of Dof in the past,its regulatory network involving in plant leaf growth and development is not perfect,and there is still great research potential.In the previous gene function research,our research group found that some Dof family members in Medicago truncatula affected the expression of downstream leaf development related genes,and screened MtDof32 gene through bioinformatics analysis and expression characteristics analysis.Under this background,the purpose of this study is to clone MtDof32 gene from Medicago truncatula,study the function of this gene,and understand its molecular mechanism involving in growth and development and stress resistance regulation through protein interaction and expression analysis.In this study,we focused on MtDof32 gene cloning,bioinformatics analysis of MtDof32,gene expression pattern,subcellular localization,physiological and biochemical analysis of MtDof32 overexpression plants,and protein interaction analysis of MtDof32.The results are as follows:1.Based on the genomic information of M.truncatula,the c DNA of MtDof32 was cloned.Bioinformatics analysis showed that MtDof32 encoded 486 amino acid,this TF belong to the second subfamily of the Dof family,and promoter cis-elements of MtDof32 contain growth,development and stress related elements.2.The expression of MtDof32 was highest in leaves,16-fold higher than that in the flower,and was induced by abiotic stress（Na Cl,PEG and 4℃）,and was inhibited by exogenous hormones（ABA,GA₃,SA）.3.Overexpression of MtDof32 in Arabidopsis showed decreased branches,late flowering,increased the size of leaf and flower organ.The average number of total branches was decreased by 4.6-5,and the flowering time was 13-20 days later.Under osmotic and salt stress,the growth of transgenic and wild-type Arabidopsis plants were significantly inhibited,but the root length and fresh weight of transgenic plants were significantly higher than that of wild-type plants,indicated that overexpression of MtDof32 enhanced the tolerance of Arabidopsis to osmotic and salt stress.4.The expression levels of At DWARF27,At CCD7,At CCD8,At MAX1,At MAX2,At SMXL6,At SMXL7 and At SMXL8 genes related to the biosynthesis pathway of strigolactones were detected in MtDof32 transgenic Arabidopsis thaliana,the results showed that the expression levels of At DWARF27,At CCD7 and At CCD8 had no significant change,but the expression levels of At MAX1 and At MAX2 were significantly up-regulated,while the expression levels of At SMXL6,At SMXL7 and At SMXL8 were significantly down-regulated.These results suggest that MtDof32 may affect the synthesis or transport of strigolactones,thereby regulating plant branching.5.Overexpression of MtDof32 significantly affected the growth and development of M.truncatula.Compared with wild-type M.truncatula,the transgenic plants had larger leaves and flower organs,flowering time was 8-11 days later on average,and the number of primary branches was decreased by 1.7-1.8 on average at 45 days of growth.The results of scanning electron microscopy showed that the cell volume of transgenic M.truncatula was significantly increased.6.In order to elucidate the regulatory mechanism of MtDof32 on plant organ size,the interacting protein was screened in yeast library with MtDof32 as bait protein,and the interacting protein MtEBP1 was obtained.Subcellular localization analysis showed that both MtDof32 and MtEBP1 proteins were expressed in the nucleus.The interaction between MtDof32 and MtEBP1 proteins was verified by BIFC double molecule fluorescence complementation experiments.Overexpression of MtDof32 upregulated the expression level of MtEBP1,suggested that MtDof32 and MtEBP1 coordinately regulated the growth and development of plant organs.Our results indicated that MtDof32 was an important transcription factor regulating plant growth and development,and play a role in the resistance to abiotic stress.This study deeply analyzed the mechanism of MtDof32 in the process of plant organ growth and branching regulation,and provided a theoretical basis for the breeding of new alfalfa varieties.