| Bromodomain-containing proteins,acting as"epigenetic readers"can bind to acetylated histones and regulate the expression of numerous genes,are widely involved in the assembly of nucleosomes,activation or repression of genes,and regulation of enzyme activity.They play an important role in many biological processes such as growth,development,and stress response.However,at present,the reports on the function and mechanism of bromodomain-containing proteins mainly focus on animals,while the research reports on plant bromodomain-containing proteins are few,and the function and mechanism of most plant bromodomain-containing proteins are still unclear.Mulberry is a perennial economic tree species,has good ecological,economic and social benefits.It is an important way to realize the economic and ecological value of mulberry to excavate new resistance genes and cultivate new resistant varieties.In this study,the bromodomain-containing phosphoprotein gene（Named as MuBRD1）was cloned from mulberry and the physicochemical properties of the encoded protein were analyzed,and the phosphorylation sites and subcellular localization of MuBRD1 protein was identified.Moreover,the expression characteristics and biological functions of the MuBRD1 gene were studied.In addition,the influence of protein phosphorylation on its function and mechanism was investigated,and the upstream transcription factors regulating the MuBRD1 gene were predicted and the interacting proteins of MuBRD1 were identified.At last,the functions and mechanisms of MuBRD1 protein were discussed.The research results are expected to provide genetic resources for mulberry breeding,and also provide reference for the research on the function and mechanism of the plant bromodomain-containing protein family.The main results are as follows:（1）Cloning of the MuBRD1 gene and characterization of its encoding protein.MuBRD1gene was cloned which encodes a protein of 784 amino acids,without a signal peptide or a transmembrane domain.The MuBRD1 protein has one conserved bromodomain with other homologous proteins from other plants.In addition,it was found that MuBRD1 protein contains multiple phosphorylation sites,and the phosphorylation sites of Ser363 and Ser364were verified using Phos-tag SDS-PAGE and Western blot techniques.Subcellular localization analysis showed that the MuBRD1 protein is located in the nucleus.Mutating Ser363 and Ser364 to alanine(Mu BRDSA)or aspartic acid(Mu BRDSD)did not affect the subcellular localization of the MuBRD1 protein.（2）Analysis of the expression characteristics and biological functions of the MuBRD1gene.The MuBRD1 gene is expressed in various tissues of mulberry such as roots,stems,and leaves,but the expression level of MuBRD1 varies among different tissues.Overexpression of the MuBRD1 and Mu BRDSAgenes in Arabidopsis significantly increased the expression of flowering genes FT and SOC1,while inhibited the expression of the flowering inhibitory gene FLC,and promote early flowering in the transgenic plants.However,overexpression of the Mu BRDSDgene did not significantly affect the expression of FT,SOC1,and FLC in transgenic plants compared to wild-type plants,nor did it affect the time of flowering.In addition,it was found that the expression of the MuBRD1 gene is induced by Na Cl,and overexpression of the gene in Arabidopsis can increase the salt tolerance of transgenic plants.The simulated dephosphorylation mutation did not affect the salt tolerance of the MuBRD1gene,but the simulated phosphorylation mutation affected the salt tolerance of the gene.（3）Identification of the upstream regulatory factors of MuBRD1 gene.Yeast one-hybrid assays demonstrated that the transcription factor Mu MYB12 can bind to the promoter of the MuBRD1 gene.At the same time,transient infiltration of tobacco,GUS tissue staining,and luciferase reporter gene assays demonstrated that the Mu MYB12 protein can enhance the promoter activity of the promoter of MuBRD1 gene.（4）Prediction and identification of MuBRD1 protein-interacting proteins.Ten proteins that interact with MuBRD1 were predicted using online software provided by bioinformatics websites.Furthermore,through yeast two-hybrid and luciferase complementation assays,it was verified that the mulberry histone acetyltransferase（Mu MYST）and bromodomain-containing protein 3（Mu BRD3）can bind to the MuBRD1 protein,while simulated dephosphorylation does not affect the interaction of MuBRD1 with Mu MYST or Mu BRD3 proteins,and the MuBRD1 simulated phosphorylation can no longer bind to Mu MYST nor MuBRD3. |
