LEA家族基因参与仁用杏花期调控的分子机制

[Objective]The aim of this study was to Understand the response mechanism of regulating the flowering period of kernel-using apricots(P.armeniaca L.×P.sibirica L.)under low temperature and achieving artificial regulation of flowering period is one of the effective ways to solve the problem of low and unstable yield of kernel apricot.[Method]The predicted peptide sequences of LEA members were acquired from the genome database(unpublished)of kernel-using apricot(P.armeniaca L.×P.sibirica L.),to construct a local protein database.BLASTP searches were performed using LEA protein sequences of A.thaliana and poplar as queries.Phylogenetic analysis and collinearity analysis were carried out for LEA family members in the genomes of P.armeniaca×.sibirica,A.thaliana,and P.trichocarpa,and the chromosome distribution,gene replication events,conservative Motif,and Cis-regulatory element of PasLEAs family genes were analyzed.Based on the analysis of expression patterns of PasLEAs genes in different tissues(flower buds and stems)and different dormancy and germination periods(Physiological dormancy(PD),ecological dormancy(ED),sprouting period(SP),and germination stage(GS)),subcellular localization and Gene co-expression network analysis of important family members were carried out,and the phenotype and gene expression of PasLEAs transgenic yeast and Arabidopsis under low temperature stress,as well as Yeast two-hybrid(Y2H)assay,Yeast one-hybrid(Y1H)assay,Bimolecular fluorescence complementation assay(BiFC)were used to explored the interaction information and regulatory mechanisms of PasLEA3-2 and Pasdehydrin-3.[Result]In this study,54 LEA members were genome-wide identified from the kernel-using apricots(P.armeniaca L.×P.sibirica L.)genome and clustered into eight subgroups according to phylogenetic relationship.A total of 14 collinear blocks containing LEA genes between P.armeniaca×P.sibirica and Arabidopsis thaliana were observed by synteny analysis,and 9 collinear blocks were identified between P.armeniaca and poplar.Gene structure and conserved motif composition analyses showed that a conserved exon-intron pattern and conserved motifs are exist within members of each subgroup.Whole-genome duplication(WGD),segmental duplication and tandem duplication are likely played an vitrol role in the gene cluster and duplication of the LEA family in P.armeniaca×P.sibirica.Expression analysis using both RNA-seq data and quantitative real-time RT-PCR(qRT-PCR)showed that PasLEA2-20,PasLEA3-2,PasLEA6-1,Pasdehydrin-3 and Pasdehydrin-5 were induced in flower buds during the dormancy and sprouting periods of P.armeniaca×P.sibirica.A PasLEA coexpression network showed that PasLEAs were coexpressed with 15 cold resistance genes or bud dormancy/dormancy release related genes such as PasEREBP,PasSer/Thr and PasWRKY.The expression pattern of all of the PasLEAs coexpressed with cold stress-associated genes was consistent with the four periods(Physiological dormancy(PD),ecological dormancy(ED),sprouting period(SP),and germination stage(GS))of flower buds of P.armeniaca×P.sibirica.An A.thaliana ortholog-based protein-protein interaction network was constructed,combined yeast two-hybrid(Y2H)and BiFC experiments revealed that PasLEA proteins had strong connections with the cold resistance pathway,such as PasLEA1-2 could interact with PasERD4,PasLEA3-1 could interact with PasXTH9,PasSMP-3 could interact with PasEXPA22,and PasDehydrin-3 could interact with PasCBF1.Remarkably,overexpression of PasLEA3-2 and Pasdehydrin-3 accerated the protein accumulation in yeast in low temperature.PasLEA3-2 and Pasdehydrin-3 overexpressed in Arabidopsis thaliana in cold stress further revealed extended pod length,shortened the bolting time and flowering time,improved survival rate and seed setting rate,and increased accumulation of proline and antioxidative enzyme activities,and a higher expression of flower development associated genes such as AtAP1,AtBTF3L,AtEBF1-1,AtLFY,AtPUB26,AtGl and AtRAV1 compared with wild-type plants.Yeast one-hybrid(Y1H)assay confirmed PasGBF4 and PasDOF3.5 can serve as upstream regulatory factors bind to the promoter of PasLEA3-2,and PasDOF2.4,PasDnaJ2 and PasAP2 can bind to Pasdehydrin-3 promoter to regulate the expression of downstream genes.[Conclusion]Overall,this study presents a comprehensive overview of evolutionary relationship,protein interaction,and functional analysis of PasLEA genes during dormancy and sprouting periods of P.armeniaca×P.sibirica,and identifies candidate genes for improving cold resistance and manipulated flower bud dormancy in P.armeniaca×P.sibirica.