Cloning,Expression Of HDA9 And AGL19 And Their Mechanism In Regulating Key Flowering Integrators Of Brassica Juncea

Brassica juncea is a kind of vegetable crop which is cultivated throughout China.Flowering is a transition from vegetative to reproductive stage in B.juncea.And flowering time will greatly affect the cultivating,breeding and F1 generation of B.juncea.The histone H3 and H4 of AGL19 were affected by the deacetylation of HDA9,which determined its chromatin environment and then regulated the expression of FT gene,resulting in the delay of flowering time.However,it is still elusive that whether HDA9 and AGL19 regulate key flowering integrators of SOC1 and AGL24.In this study,BjuHDA9 and BjuAGL19 from B.juncea were cloned and their expressions were measured.Subsequently,Interactions of BjuHDA9 and BjuAGL19 with BjuSOC1 and BjuAGL24 were identified via a series of assays including yeast two-hybrid,pull-down,yeast one-hybrid and Dual-Glo?Luciferase.The results were as follows:1.Cloning and bioinformatics analysis of BjuHDA9 and BjuAGL19 gene in B.junceaIn this study,BjuHDA9 and BjuAGL19 genes were obtained from B.juncea via PCR.The cDNA sequences of BjuHDA9 and BjuAGL19 were 1284bp and 666bp,respectively.BjuHDA9 encoded 426 amino acids with the predicted molecular weight of 48.73kDa and the theoretical pI of 5.26.BjuAGL19 encoded 221 amino acids with the predicted molecular weight of 25.16 kDa and the theoretical pI of 8.94.2.The expression patterns of BjuHDA9 and BjuAGL19 genesThe BjuHDA9 and BjuAGL19 genes were respectively expressed in all five tissues including root,stem,leaf,flower and flower bud in B.juncea.BjuHDA9 was mainly expressed in leaves,with relatively few expressions in the stem.BjuAGL19 expressed at the the highest level in roots.Furthermore,the expression of BjuHDA9 was induced by short-day photoperiod,but inhibited by low-temperature vernalization.3.Protein-protein interactions of BjuHDA9 with BjuSOC1 and BjuAGL24The yeast two-hybrid assays showed that there were no protein interactions of BjuHDA9with BjuSOC1 and BjuAGL24.And the results were validated via the pull-down assays.4.Protein-protein interactions of BjuAGL19 with BjuSOC1 and BjuAGL24The yeast two-hybrid assays showed that there was no interaction of BjuAGL19 with BjuSOC1,but there was protein interaction of BjuAGL19 with BjuAGL24.The results were verified via the pull-down assays.5.Protein-DNA interactions of BjuHDA9 with promoters of BjuSOC1 and BjuAGL24The yeast one-hybrid yeast assays indicated that Y1HGold[（pAbAi-BjuSOC1）×（pGADT7-BjuHDA9）]could grow normal on SD/Leu/AbA¹⁰⁰ plate,and Y1HGold[（pAbAi-BjuAGL24）×（pGADT7-BjuHDA9）]could grow on SD/Leu/AbA³⁵⁰.It suggested that HDA9 interacted with promoters of BjuSOC1 and BjuAGL24.The above results were verified by Dual-Glo?Luciferase Assays.Furthermore,the specific fragments of BjuSOC1 and BjuAGL24 interacting with BjuHDA9 were identified via yeast one-hybrid and Dual-Glo?Luciferase.And the results demonstrated that BjuHDA9 interacted with BjuSOC1-3 and BjuAGL24-3 in vitro and in vivo.6.Protein-DNA interactions of BjuAGL19 with promoters of BjuSOC1 and BjuAGL24The yeast one-hybrid yeast indicated that Y1HGold[（pAbAi-BjuSOC1）×（pGADT7-BjuAGL19）]could not grow normal on SD/Leu/AbA¹⁰⁰ plate,and Y1HGold[（pAbAi-BjuAGL24）×（pGADT7-BjuAGL19）]could not grow on SD/Leu/AbA³5050 plate.It indicated that BjuAGL19 could not interact with promoters of BjuSOC1 and BjuAGL24.The above results were validated via Dual-Glo?Luciferase assays.7.Mutants of BjuHDA9 interact with BjuSOC1 and BjuAGL24The 172th,174th and 261th in BjuHDA9 were respectively mutated by overlap extension PCR.Yeast one-hybrid and Dual-Glo?Luciferase assays found that BjuHDA9mutants still interacted with BjuSOC1 and BjuAGL24,and the interaction strength was reduced.