Interactions Of HDA9 And AGL18 With Flowering Integrator Factors AGL19, AGL24 And SOC1 In Brassica Oleracea Var. Italica

The floret is main commercial organ of Brassica oleracea var.italica,so timely flowering has a great impact on yield and quality.Flowering represents the transition from vegetative growth to reproductive growth.Accurate control of this process requires a precise regulatory network in plant.Previous studies have confirmed that many members of the MADS-box transcription factor family in Arabidopsis are involved in flowering regulation,such as AGL18?AGL19?AGL24 and SOC1.At the same time,AGL18?or AGL15?can prevent early flowering by recruiting HDAC complexes to FT chromosome loci for cyclic histone deacetylation modification.TPL and SAP18,transcriptional co-inhibitors involved in regulating flowering time,can interact with EAR motifs.The AGL18 cloned in this experiment contains an EAR motif.It remains unknown whether they can regulate each other or not.In order to elucidate the regulatory relationships between proteins,we cloned HDA9,AGL18,SAP18 and TPL genes from Brassica oleracea var.italica.The protein-protein interactions of HDA9,AGL18,SAP18 and TPL with flowering integrators AGL19,AGL24and SOC1 were detected via Yeast Two-Hybrid system and GST pull-down.The interaction relationships of HDA9,AGL18,AGL19,SAP18 and TPL with AGL24 and SOC1 promoters were detected by Yeast One-Hybrid system and Dual-Glo^?Luciferase system.This study lays a foundation for further study on the flowering regulation mechanism in Brassica oleracea var.italica.The main results are as follows:1.Cloning and Bioinformatics Analysis of HDA9,AGL18,SAP18 and TPL in Brassica oleracea var.italica.In this study,HDA9,AGL18,SAP18 and TPL of Brassica oleracea var.italica were cloned.Sequence-length of the cDNAs were 1281 bp,777 bp,459 bp and 3452 bp,encoding426,258,152 and 1129 amino acids,respectively.The molecular weights of the corresponding proteins were 48.75 kD,28.73 kD,17.36 kD and 124.33 kD.They were all hydrophilic proteins and closely related to Brassica oleracea var.oleracea and Brassica napus,followed by Arabidopsis.2.AGL18 interact with AGL24 in Brassica oleracea var.italica.Using Y187?pGADT7-AGL18?as prey protein and Y2HGold?pGBKT7-AGL24?as bait protein,Yeast Two-Hybrid system showed that the hybrid yeast strain could grow normally on DDO/A and QDO media,and grew blue on QDO/X/A.The result of vector exchange was consistent.GST pull-down found that lane of GST-AGL18 and HIS-AGL24co-incubation had two protein bands,the sizes were 55 kD and 45 kD respectively,consistently with the molecular-weight of GST-AGL18 and HIS-AGL24,indicating that AGL18 could interact with AGL24 protein.3.AGL18,HDA9,SAP18 and TPL interact with SOC1 promoter in Brassica oleracea var.italica.Yeast One-Hybrid system showed that Y1HGold[?pAbAi-SOC1?×?pGADT7-AGL18?],Y1HGold[?pAbAi-SOC1?×?pGADT7-HDA9?],Y1HGold[?pAbAi-SOC1?×?pGADT7-SAP18?],and Y1HGold[?pAbAi-SOC1?×?pGADT7-TPL?]strains could grow normally on SD/-Leu/ABA¹⁰⁰solid plates.It suggested that AGL18,HDA9,SAP18 and TPL protein could interact with SOC1 promoter.The results of Dual-Glo^?Luciferase system showed that AGL18,HDA9,SAP18 and TPL could interact with SOC1 promoter in Nicotiam.benthamiana.4.AGL18 and HDA9 interact with AGL24 promoter in Brassica oleracea var.italica.The interactions of AGL18 and HDA9 with AGL24 promoter in Brassica oleracea var.italica were confirmed via Yeast One-Hybrid system and Dual-Glo^?Luciferase system.5.HDA9 could not interact with AGL18,AGL19,AGL24 and SOC1 in Brassica oleracea var.italica.The interaction relationships of HDA9 with AGL18,AGL19,AGL24 and SOC1 were detected by Yeast Two-Hybrid system.The results showed that the hybrid yeast strains could not grow normally on DDO/A,QDO and QDO/X/A media.It means that HDA9 could not bind to AGL18,AGL19,AGL24 and SOC1.Further,GST pull-down showed that only HIS-HDA9 protein band was found in the protein co-incubation lanes,indicating that HDA9could not interacted with AGL18,AGL19,AGL24 and SOC1.6.SAP18 could not interact with HDA9,AGL18,AGL19,AGL24 and SOC1 in Brassica oleracea var.italica.The interaction relationships of SAP18 with HDA9,AGL18,AGL19,AGL24 and SOC1proteins were identified by Yeast Two-Hybrid system.The results showed that diploid fused yeasts could not activate the expression of reporter genes HIS3,MEL1,AUR1-C and ADE2.GST pull-down showed that the co-incubation lanes of HIS-SAP18 with GST-HDA9,GST-AGL18,GST-AGL19,GST-AGL24 and GST-SOC1 had only one protein band of about37 kD,indicating that SAP18 could not interacted with HDA9,AGL18,AGL19,AGL24 and SOC1.7.TPL could not interact with SAP18,HDA9,AGL18,AGL19,AGL24 and SOC1 in Brassica oleracea var.italica.The Y187?pGADT7-TPL?was prey protein,Y2HGold?pGBKT7-SAP18?,Y2HGold?pGBKT7-HDA9?,Y2HGold?pGBKT7-AGL18?,Y2HGold?pGBKT7-AGL19?,Y2HGold?pGBKT7-AGL24?and Y2HGold?pGBKT7-SOC1?as bait proteins.Yeast Two-Hybrid system showed that TPL could not interact with SAP18,HDA9,AGL18,AGL19,AGL24 and SOC1.Vectors interchanged,the results were consistent.