Function And Molecular Mechanism Of MdNF-YB18 In Regulating The Floral Induction

In Arabidopsis,nuclear factor Y(NF-Y)transcription factor can participate in flower formation regulation with other protein by forming complexes.The flower formation characteristics of apple and other woody plants are significantly different from those of Arabidopsis and other model plants,but the relevant mechanism remains to be elucidated.In this study,we chose ‘Nagafu No.2' as the material,and used experimental methods such as bioinformatics analysis,Yeast Two-Hybrid assay,bimolecular fluorescence complementary and double luciferase assay to systematically study the mechanism of Md NF-YB18 in the cooperative regulation of Md FT transcription.The main research results are summarized as follows:1.Identification,evolution,expression characteristics and functional analysis of Md NF-Ys family members in apple.(1)51 members of the Md NF-Ys family were identified in the new genome of Apple,including 11 Md NF-YAs,26 Md NF-YBs and 14 Md NF-YCs.(2)The results of evolution and structural analysis of Md NF-Ys family showed that the gene and protein structures of each subfamily members were highly conserved,and the differences among subfamilies were obvious.The results of promoter cis-acting element analysis showed that they have the acting elements which are in response to ABA,salicylic acid,gibberellin,light signal and biological clock,while gibberellin,light signal and biological clock can play important roles in regulating flower formation,indicating that the family members may play a functional role in apple floral induction by participating in the above regulatory pathways.(3)In the analysis of expression characteristics of Md NF-Ys family,we found that 51 genes had relatively high expression in flower,leaf and fruit tissues,and Md NF-YBs subfamily members had relatively high expression in flower tissues;and Md NF-YB5,Md NF-YB18,Md NF-YC3 and Md NF-YC7 have higher differential expressions in 'Nagafu No.2' and 'Qinguan'.Combined with the previous research of our team,Md NF-YB18 was selected as the key gene in regulating flowering,and the 35S: Md NF-YB18 overexpression vector was constructed for the transgenic Arabidopsis,overexpression lines showed strongly early flower characteristics,suggesting that Md NF-YB18 can promote flowering in Arabidopsis..2.Screening,validation and regulatory mechanism analysis of Md NF-YB18 interaction proteins.(1)Md NF-YB18 was as a bait protein and the yeast two-hybrid technology was used to screen for the interactive proteins in the flower bud library.A total of 148 interactive proteins were screened,which were respectively related to glycolytic pathway,photoperiod pathway,heat shock proteins and stress resistance.(2)The proteins related to flower formation were selected for interaction verification,including Md NF-YC3,Md NF-YC7,Md NF-YC14,Md14-3-3 protein,Md F-box protein,Mdb LHP1-Like,Md MYB62.The results showed that Md NF-YB18 can interact with Md MYB62,Md NF-YC3,Md NF-YC7 and Md NF-YC14.And results of double molecule fluorescence complementary test further showed that NF-YB/NF-YC dimer complex could be formed in apple.(3)By constructing Mdp FT-0800 LUC vector and Md NFYB18-62 SK vector for the dual-luciferase reporter assays,Md NF-YB18 can activate the promoter of Md FT,which showed that Md NF-YB18 can directly regulate the transcription and expression of Md FT.In summary,Md NF-YB18 can promote the flowering of Arabidopsis thaliana,and it plays a significant role by activating Md FT.Md NF-YB18 can interact with Md NF-YC3,Md NF-YC7 and Md NF-YC14 to form a dimer complex.Combined with the preliminary research by our team,there may be Md COL/Md NF-YB/Md NF-YC--Md FT regulation mode involved in floral induction in apple.