Identification And Functional Analysis Of Pear Dormancy Associated DAM Genes

MADS-box transcription factor family is one of the well-studied gene families in plants.It plays an important role in the biological processes such as flower induction,floral organ development,dormancy and fruit development.DAM genes belong to the SVP clade,which is represented in Arabidopsis thaliana by SVP and AGL24.DAM genes have been suggested directly associated with bud dormancy regulation.They have been isolated and studies in different deciduous fruit trees such as peach,pear,apple,plum and kiwifruit.Although three pear DAM genes have been obtained through homologous cloning,the research on them mainly focused on their expression pattern analysis but lacked functional verification,so it is necessary to conduct a more comprehensive study on DAM genes in pear.In this study,five pear DAM genes were obtained,and their phylogenetic analysis,expression analysis and functional verification were performed.The main results are as follows:1.Identification of pear MADS-box gene familyA total of 100 MADS-box gene family members were identified in the'Dangshansuli' genome,which can be divided into 17 clades by phylogenetic analysis.Collinearity analysis revealed that the whole genome duplication(WGD)events that pears experienced 30-45 million years ago promoted the expansion of the MADS-box gene family members.Expression analysis showed that different MADS-box genes have different expression patterns in pear fruit development and pear bud dormancy,which might play different regulatory roles in these processes.2.Identification of pear DAM genesFive DAM genes were identified in the 'Dangshansuli' genome,namely PpyMADS28,PpyMADS29,PpyMADS30,PpyMADS31 and PpyMADS71.Protein interaction analysis revealed that there was interaction between the remaining DAM proteins except for the PpyMADS30 protein.The qRT-PCR analysis showed that the expression patterns of pear DAM genes were similar in the high-chill cultivar'Dangshansuli' and low-chill cultivar 'Cuiguan',but they significantly changed in the high-chill variety.The expression of PpyMADS28,PpyMADS29 and PpyMADS71 during the dormancy process of pear buds increased continuously,and the expression decreased sharply after endodormancy release.PpyMADS30 and PpyMADS31 were down-regulated after the pear buds entering endodormancy.In addition,PpyMADS28,PpyMADS29 and PpyMADS31 had higher expression in stamens during flowering of'Xueqing'3.Overexpression of Pear DAM Genes in ArabidopsisTransgenic lines of DAM genes were obtained by floral-dip method.Overexpression of PpyMADS28,PpyMADS29 and PpyMADS31 genes in Arabidopsis caused sepals developed into leaf-like structures,and these leaf-like sepals did not abscised from the silique after flowering.Also,these genes overexpression can reduce fertility.However,overexpression of PpyMADS30 and PpyMADS71 genes did not affect flower development and fertility in Arabidopsis,indicating that the DAM genes have functional differentiation.Overexpression of DAM genes did not affect Arabidopsis seed germination.4.Study on alternative splicing of pear DAM genesIt was found by cloning that PpyMADS31 and PpyMADS71 genes had alternative splicing.Three PpyMADS31 splice isoforms and four PpyMADS71 splice isoforms were identified from 'Dangshansuli'.Differences in protein structure among different splice isoforms affect the interaction between proteins.Overexpression of the three splice isoforms of PpyMADS31 gene in Arabidopsis led to different phenotypes,indicating that the structure difference of the PpyMADS31 splice isoform resulted in functional differentiation.No significant differences were observed among overexpression Arabidopsis lines of the four splice isoforms of the PpyMADS71 gene.In summary,five DAM genes were identified in the pear genome.Among them,PpyMADS28,PpyMADS29,PpyMADS31 and PpyMADS71 genes were potentially involved in the regulation of pear dormancy.PpyMADS28,PpyMADS29 and PpyMADS31 genes were propably involved in floral organ development during flowering.In addition,alternative splicing isoforms of PpyMADS31 and PpyMADS71 were identified,and there are differences in the functions of different splice isoforms.This study preliminarily revealed the evolution and functional differentiation of the pear DAM genes,laying the foundation for our further research of the function and molecular regulatory network.However,the functional analysis of this study using heterologous Arabidopsis as a system could not fully reveal the function of pear DAM genes and requires homologous transgene validation in the future.