Cloning And Functional Study Of Passiflora Edulis Flowering Genes PeFt,PeSVP And PeAGL24

Passion fruit(Passiflora edulis Sims)is a perennial evergreen vine of the Passifloraceae genus Passiflora,and its cultivation area has been expanding in recent years.Passion fruit is a tropical and subtropical fruit tree,but when the temperature is too high,there will be poor flowering or no flower phenomenon,which seriously affects the yield.PEBP family and MADS-box family transcription factors play crucial roles in this flowering process.Among them,FT was the flowering promoting factor.SVP was used as a flowering suppressor.However,AGL24 could promote flowering.In this paper,the function and interaction of FT,SVP and AGL24 in Passiflora edulis,which lay the foundation for further revealing the molecular regulation network flower-forming,and can be applied to molecular breeding research to promote the early flowering.The main experimental results were as follows:1.Two FT genes,named PeFT1 and PeFT2,were identified in the Passiflora edulis genome.Two SVP gene sequences and one AGL24 gene sequence were identified,named PeSVP1,PeSVP2 and PeAGL24,respectively.The prediction results of the secondary structure of PeFT1 and PeFT2 proteins showed that the random coil and extended chain accounted for a large proportion,suggesting that this region plays an important role in binding to the downstream FD to regulate flowering.Protein domain analysis of PeSVP1,PeSVP2,and PeAGL24 revealed that all of them have two relatively conserved domains,MADS-box and K-box.By establishing a three-level model of the protein structure,it was found that α-helices and random coils accounted for a large proportion of the protein.Based on transmembrane protein and signal peptide prediction,PeFT1,PeFT2,PeSVP1,PeSVP2 and PeAGL24 proteins were predicted to be non-secreted proteins because they did not have transmembrane structure and signal peptide.By subcellular localization prediction,it is predicted that PeFT1,PeFT2,PeSVP1,PeSVP2,and PeAGL24 proteins localized to the nucleus.2.Two PeFT genes,two PeSVP genes and one PeAGL24 gene were cloned.The CDS region length of the PeFT1 gene is 587 bp,the CDS region length of the PeFT2 gene is 525 bp,the CDS region length of the PeSVP1 gene is 681 bp,the CDS region length of the PeSVP2 gene is654 bp,and the CDS region length of the PeAGL24 gene is 708 bp.Fusion-expressed proteins were constructed,and the proteins encoded by PeFT1,PeFT2,PeSVP1,PeSVP2 and PeAGL24 genes were all localized in the nucleus to play a physiological role in the nucleus.3.Real-time fluorescence quantitative technology was used to detect the expression specificity of PeFT gene in different tissues.The expression of both PeFT genes are tissue-specific and low in reproductive organs.The expression level was relatively high in mature vegetative organs,and almost no expression were found in young vegetative organ terminal buds.Among the different temperature treatments,the highest expression of the two PeFT genes were observed at 33 ℃,and the flowering regulation was mainly regulated by the PeFT2 gene.The expression of both PeSVP genes were significantly up-regulated at lower temperature(28 ℃).PeSVP1 was more sensitive to temperature than PeSVP2.The relative expression of PeAGL24 gene was greatly affected by the change of temperature,and the expression was higher at the lower temperature(28 ℃).The flowering was mainly regulated by PeFT2.4.When PeFT2 and PeAGL24 genes were overexpressed in Arabidopsis thaliana,transgenic Arabidopsis blooms 3-5 days earlier than wild type.And at flowering,rosettes have 5 to 7 fewer leaves than wild-type Arabidopsis.The PeFT2 gene and PeAGL24 gene promoted the expression of flowering related genes such as FT and SOC1 in Arabidopsis thaliana.The PeFT2 gene and PeAGL24 gene play an important role in the regulation of Passiflora edulis flowering.5.The interaction between PeSVP1,PeSVP2 and PeAGL24 transcription factors and PeFT2 promoter was verified by yeast single hybrid experiment.The yeast single hybrid assay showed that PeSVP1 and PeSVP2 could interact with the promoter of PeFT2 gene.Further analysis of the dual luciferase transient expression data showed that PeSVP1 and PeSVP2 had a significant inhibitory effect on the promoter of PeFT2 gene.PeAGL24 could interact with the promoter of PeFT2 gene.Further analysis of dual luciferase transient expression data showed that PeAGL24 had a significant activation effect on the PeFT2 gene promoter.