Functional Identification Of Brassica Juncea EBS Gene And Its Interaction With Flowering Integrator

Brassica plants contain a variety of oil crops and vegetable crops that are essential to human nutrition,the flowering time greatly affects the yield and quality of crop product organs.Brassica juncea is an important cruciferous vegetable crop in the Brassicaceae family.Timely flowering can ensure the normal growth of plants and a successful transition from vegetative to reproductive stages.At present,there are many ways to control the flowering of Brassica juncea,such as vernalization pathway,photoperiod pathway,gibberellin pathway,autonomous pathway,age pathway and endogenous pathway.There are no separate flowering inhibition pathways,but flowering inhibitors can regulate flowering by interacting with flower promoters and flowering signal integrators,which is essential to prevent premature flowering.EARLY BOLTING IN SHORT DAYS（EBS）is the protein with related chromatin remodeling factors.In Arabidopsis,it acts as a transcriptional repressor,which can suppress the expression of FT gene and regulate flowering time.EBS protein is enriched in Arabidopsis SOC1,EMF1 and FLC.The flowering time is regulated by identifying two histone modifications with opposite functions and changing their binding preferences.In addition,Arabidopsis EBS is also involved in the regulation of other MADS-box transcription factors,such as APETALA3（AP3）,PISTILLATA（PI）and AGAMOUS（AG）,which affect the floral organ development of Arabidopsis.EBS can also interact with AGAMOUS-LIKE67（AGL67）to regulate Arabidopsis seed dormancy.However,what is the function of EBS in Brassica juncea?Is it possible to adjust the flowering time by directly interacting with flowering integrators（such as SOC1,AGL24）?It is still unclear and needs to further study.In this study,we cloned the mustard EBS（BjuEBS）gene and analyzed its function in flowering time control.Using multiple assays of yeast two-hybrid,GST pull-down,yeast one-hybrid and Dual-Glo?luciferase,the interactions between EBS and flowering integrators were detected,respectively.The experimental results are as follows:1.Molecular cloning and sequence analysis of BjuEBS gene in Brassica juncea.The BjuEBS gene of c DNA sequence in Brassica juncea was cloned by PCR.It contained 675bp and encoded 224 amino acids.The predicted molecular weight of BjuEBS protein was 25.5 k Da.NCBI online analysis showed that the amino acid sequences of BjuEBS was highly similar to Ath EBS in Arabidopsis.2.Photoperiod pathway regulated BjuEBS expression in Brassica juncea.In order to explore the dynamic expression pattern of BjuEBS in different organs and tissues of Brassica juncea under long-day photoperiod conditions,the RNA was extracted from the yong leaves every 20 days for q RT-PCR.The results showed that after long-day treatment,the expression of BjuEBS in the roots,stems and leaves of Brassica juncea showed a downward trend.However,with the prolonged treatment of long-day photoperiod,the expression level of BjuEBS gradually decreased.When treated for 40 days,the mustard plants approached to flowering.At this time,BjuEBS highly expressed in the shoot tips and leaves,but slightly expressed in the roots.The results indicated that the long daylight treatment could inhibit the expression of BjuEBS in Brassica juncea.3.BjuEBS ectopic overexpression in Arabidopsis resulted in earlier flowering.The BjuEBS gene of Brassica juncea was recombined into p Bin35SRed3 vector to construct p Bin35SRed3-BjuEBS fusion plasmid,which was then transformed into Arabidopsis thaliana using Agrobacterium-mediated method.The non-transgenic plants were used as controls for long-day light treatment.The results showed that the p Bin35SRed3-BjuEBS transformed plants showed earlier inflorescences and flower buds,compared with the control plants.This implied that ecotopic overexpression of BjuEBS in Arabidopsis thaliana promoted early flowering under the LD condition.It suggested that the appropriate amount of BjuEBS was very important to maintain its flowering time.4.BjuEBS protein could not interact with BjuSVP,BjuSOC1 or BjuAGL24 protein.Brassica juncea BjuEBS is involved in flowering time control,but it is unclear whether BjuEBS regulates flowering time by interacting with BjuSVP,BjuSOC1 and BjuAGL24 proteins.Using yeast two-hybrid research,we found that the yeast strains of BjuEBS with BjuSVP,BjuSOC1,or BjuAGL24 could not grow on SD/-Ade/-His/-Leu/-Trp/X-α-Gal media.It indicated that BjuEBS could not interact with BjuSVP,ju SOC1 or BjuAGL24 proteins.In addition,pull-down assays showed that His-tagged BjuEBS protein failed to combine with GST-tagged BjuSVP,BjuSOC1 or BjuAGL24 protein in vitro.The consistent results were obtained from the different methods above mentioned.5.BjuEBS protein interacted with promoters of BjuSOC1 and BjuAGL24.To determine whether BjuEBS directly regulated flowering by interacting with the promoters of flowering integrators,the yeast one-hybrid assay and Dual-Glo^?luciferase assay were used in this study.Yeast one-hybrid assay indicated that yeast fusion strains of BjuEBS and BjuSOC1 promoter grew normally on SD/-Leu/A¹⁰⁰medium.Similarly,yeast strains of BjuEBS and BjuAGL24promoter also grew normally on SD/-Leu/A³⁵⁰medium.It suggested that the BjuSOC1 and BjuAGL24 promoters interacted with BjuEBS protein directly.Furthermore,the Dual-Glo^?dual luciferase assays showed that the LUC/REN enzyme activity ratios of BjuEBS-BjuSOC1 or BjuEBS-BjuAGL24 were significantly higher than the negative controls,suggesting that BjuEBS could indeed bind to promoters of SOC1 and AGL24 in vivo.Subsequently,a series of truncated BjuSOC1 and BjuAGL24 promoters were constructed for screening their interaction regions.The results showed that the BjuSOC1-1 and BjuAGL24-2 were the key truncated forms and target regions to bind BjuEBS protein.