The Molecular Regulation Mechanism Of Bud Dormancy Release In Prunus Mume

Prunus mume Sieb.et Zucc.is a traditional Chinese flower,with high ornamental value and rich cultural connotation.P.mume can be flowering in relatively low temperatures in early spring.Studies have indicated that this is related to bud dormancy release process.Dormancy is a self-protection mechanism for perennial plant.It is a very complex physiological and biochemical process and is regulated jointly by the external environment and internal physiology.Previous studies have indicated that hormones and sugars play crucial roles in bud dormancy release process.GAMYB and a-amylase are key genes in the interaction between hormone and sugar.However,the molecular regulation mechanism of flower bud dormancy is not yet clear.In this study,P.mume 'Zao lve' was used as experimental material.According bud flush rate at artificial conditions,four dormancy stages were identified.High-throughput sequencing technology was used to take RNA-Seq,small RNA and degradome analysis.In addition,a series of physiological and biochemical and molecular biological methods,such as yeast two-hybrid assays,bimolecular fluorescence complementation(BiFC)assays,yeast one-hybrid assays,were used to build molecular regulation model of bud dormancy release in P.mume.The main results are as follows:(1)According to the flush rate and chilling accumulation,we termed four dormancy stages:EDI(with 0%flush rate),EDII(with 45%flush rate),EDIII(with 90%flush rate)and NF(natural flush).During the dormancy release process,the ABA content continued to drop significantly,and GA1,GA3,GA4,and IAA decreased during the period from the EDI to the EDII,then increased significantly at,and the ABA/GA3 ratio continued to decrease significantly.The starch content continued to drop significantly,and the soluble total sugar,glucose,and sucrose content increased significantly.(2)The transcriptomes of the four dormancy stages identified 5,83 1 significant different expressed genes.A total of 259 known miRNAs and 298 novel miRNAs were identified at the four dormancy stages.Combining mRNA data,80 known miRNAs and 124 novel miRNAs can find their target genes.Degradome results revealed 231 cleave sites between 89 miRNAs and 205 target mRNAs,in which 45 known miRNAs,121 target genes and 132 cleave sites were identified.The results of 5'RACE indicated that Pm016605 could be cleaved by Pm-mi159a and Pm-miR319a-3p during the dormancy release process.(3)Two highly conserved GAMYB genes were found in the P.mume genome.PmGAMYB2 exhibited high expression level at EDI to EDIII stages,and then gradually decreased as dormancy release progressed;while PmGAMYBl showed extremely low level at endo-dorrmancy stages and high level at NF stage.GA3 and ABA can promote and inhibit their expression.Subcellular localization showed that PmGAMYBs protein were mainly expressed in the nucleus.Two PmGAMYBs were not toxic,but PmGAMYB1 showed auto activation.The results of yeast two-hybrid assays and BiFC assays suggested that PmGAMYB2 could form homodimers,and PmGAMYB2 could dimerize with PmGAMYB1 to form heterdimers.(4)Seven highly conserved PmAMYs were identified in P.mume genome,of which four sequences contained GARE,MBS,TATA and TC domains at their upstream promoters.PmAMY proteins are mainly distributed on plastids(chloroplast).The promoters of the PmAMYl,PmAMY3,PmAMY6 and PmAMY7 genes are all have activity.PmGAMYB2 could bind to the GARE(TAACAAA),not the mutation of this motif(TTTTAAA),in the promoter of PmAMYs.In conclusion,this study firstly integrated mRNA,miRNA,and degradome to analyze the mechanism of bud dormancy release in P.mume.Combined with a series of biochemical and molecular biological experiments,I proposed a hypothetical model for understanding the molecular regulation mechanism of bud dormancy release in P.mume.