The Vernalization Response Of BcMAF2 And Interaction Proteins Screeing Of BcFT In Non-heading Chinese Cabbage

Blossom is the conversion of plants from vegetative growth stage to reproductive growth,which is an important developmental stage during the life cycle of plants.The transition to flower is timed by endogenous and environmental signals.To study the mechanisms of flowering has important economic value for the production and breeding of vegetable crops.At present,six flowering pathways have been identified.Genes related flowering constituted a complex regulatory network,different flowering pathways integrated and activated the 'flowering response elements',FT is one of the 'flowering response elements'.In this study,we explored the vernalization response of BcMAF2 gene in non-heading Chinese cabbage,and conducted yeast two-hybrid assays to screen the proteins that interacted with BcFT.The main results are as follows:1.BcMAF2 gene which obtained from the non-heading Chinese cabbage 'Su zhou qing' encodes a protein of 192 amino acid whit PI of 6.22,a molecular weight of 21.5×103Da,has no transmembrane region.The N-terminal 1?60 amino acids of the BcMAF2 protein contains a conserved MADS-box domain and BcMAF2 belongs to the typical MADS-box protein,the identity to AtMAF2 amino acid was 72%.The subcellular localization result showed that the BcMAF2 protein was located not only in the nucleus but also in the cytoplasm and cell membrane.Material 'NHCC004' was provided to analyze the expression levels of BcMAF2 and its homologous genes at different periods of five weeks at low temperature(4 ?)with qRT-PCR:the expression level of BcMAF2 was dramatically upregulated and reached its expression peck at 14 day.With the low temperature processing,the expression of BcFLC2 decreased,but unchanged at 14?28 days.BcMAF3 gene was the most sensitive to low temperature,the expression level was significantly higher than that of the control group and reached its expression peck at 28 day.The expression levels of BcFLM and BcMAF4 showed 'low?high?low' trend and at the end of the cold treatment,the expression levels were lower than that before vernalization.2.The expression product of the bait vector pGBKT7-BcMAF2 was confirmed no autoactivation and toxicity.BcMAF2 and BcSVP interacted in vitro and in vivo.we found BcMAF2 also interacted with BcFLC2 and BcMAF4 could form the homodimer.In Arabidopsis thaliana,AtMAF3 interacted with AtFLM?AtMAF4 and AtFLC,respectively,while there were no such phenomenons in non-heading Chinese cabbage,BcMAF3 protein may have some chemical modifications or mutations in the key amino acid sites that affecting protein interactions and further studies were needed to answer this question.BcSVP can interact with other proteins other than BcMAF3,and can form homologous dimers.3.We constructed the subcellular localization vector pEZS-NL-BcFT and analysed the spatial expression of FT by subcellular localization technique.BcFT protein was located in all components of cell.We also constructed pGBKT7-BcFT bait vector,transformed into yeast strain Y2H Gold to verify self-activation and autotoxicity.The expressed product of the bait vector pGBKT7-BcFT was confirmed no autoactivation and toxicity.When screening the yeast two hybrid cDNA library of non-heading Chinese cabbage,we found two proteins that physically interacted with BcFT.Bioinformatics analysis predicted that these two proteins are hydrophilic proteins and have no signal peptide.BcPPD5 protein has a transmembrane region,in the secondary structure,the largest is the irregular crimps,there is a PsbP domain between the 163 and 296 amino acid residues.For BcVHA-E1,the largest secondary structure is the alpha helix,there is no transmembrane region and specific domain.