The Role Of MiR159 In The Regulation Of Flower Bud Dormancy Of Pear By GA

Pear belongs to the temperate deciduous fruit tree of Pyrus L.of Rosaceae and Amygdaloideae,In order to adapt to the adverse environment in winter,it has the biological characteristics of winter dormancy.When entering internal dormancy,it is necessary to accumulate a certain amount of low temperature accumulation in order to release dormancy.GA is an important substance for plants to break flower bud dormancy and promote flower bud germination,and miR159 is a key regulatory factor in plant gibberellin signal transduction pathway.Sand pear,as the main variety in Fujian Province,has the advantage of early maturity,early listing,and occupies an important position in the market,which has brought great economic benefits to the development of pear industry in Fujian.Due to the problems caused by climate change in recent years,such as returning green in autumn and winter and lack of low temperature in winter,the development of sand pear industry is greatly restricted.Therefore,the relevant molecular mechanism of pear flower bud dormancy process is explored.It is of great significance to the development of Fujian pear industry.In this paper,the short low temperature pear variety’Mixue’was used to study the role of miR159 and its target gene GAMYB in the dormancy of pear flower buds.The main results are as follows:1.Through the analysis of miRNAs transcriptome data in different periods of pear flower bud dormancy in the early laboratory,it was found that the expression abundance of miR159 family was the highest in the process of pear flower bud dormancy.It was speculated that miR159 family may play an important role in the regulation of pear flower bud dormancy,and two miR159 mature bodies,Pp-miR159a and Pp-miR159b,were screened and identified.In this paper,exogenous GA₃ was used to treat pear dormant flower buds,and fluorescence quantitative PCR was used to study the expression patterns of miR159 and target genes during pear flower bud dormancy.The results showed that the expression of Pp-miR159a and Pp-miR159b in GA₃ treatment group was generally lower than that in clear water control group,indicating that GA may play a role by down-regulating the expression of miR159.In clear water control group and GA treatment group,the expression of miR159 family was negatively correlated with the expression of predictive target gene PpGAMYB.The results of this study show that GA plays an important regulatory role in the process of pear flower bud dormancy release,probably by down-regulating the expression of Pp-miR159a,and Pp-miR159a negatively regulates the expression of target gene PpGAMYB.2.A MYB transcription factor PpGAMYB gene in gibberellin signal transduction pathway in pear flower bud was amplified from c DNA of’Mixue’pear flower bud.The CDS length of the gene is 1647bp,encoding548 amino acids.The protein sequence has the unique R₂R₃DNA binding domain and conservative elements Box1,Box2 and Box3 of the GAMYB family.Subcellular localization showed that the PpGAMYB gene was located in the nucleus.The analysis of PpGAMYB promoter showed that there were a large number of hormone response elements,light response elements,defense and stress response elements,anaerobic induction elements,low temperature induction elements and MYBHv1 binding sites and other related elements in the promoter region.The results of this study showed that PpGAMYB gene of’Mixue’pear was involved in many kinds of emergency responses such as hormone response,drought stress,cold stress,heat stress and so on.3.In order to confirm that PpGAMYB is the target gene of Pp-miR159a.The expression vectors of p1304-GFP-PpGAMYB,p1304-35S-pre-miR159a,p SK-pre-miR159a and pGreen-LUC-PpGAMYB were constructed,and the transient transformation and double luciferase interaction test of tobacco were carried out.The results of instantaneous transformation of tobacco showed that there was green fluorescence signal in the experimental group and the green fluorescence signal was significantly weaker than that in the control group,indicating that there was an inhibitory relationship between Pp-miR159a and PpGAMYB.The results of double luciferase test showed that the ratio of F-Luc to R-Luc in the test group was significantly lower than that in the control group,indicating that the enzyme activity in the test group was significantly weakened,which further indicated that Pp-miR159a could inhibit the expression of target gene PpGAMYB.The results of this study showed that Pp-miR159a negatively regulated the expression of target gene PpGAMYB and then affected the dormancy process of pear flower buds.