Molecular Mechanism Of WRKY71 And WRKY75 In Flowering Regulation Of Brassica Juncea

Brassica juncea is an allotetraploid species formed by natural hybridization between Brassica rapa and Brassica nigra,which is widely planted in the south of China.B.juncea is a long-day seed vernalization crop,and the flowering time has a great influence on its yield.Multiple factors including environmental conditions and internal genetic factors will affect the timely flowering of B.juncea.WRK Y family is one of the largest transcription factor families in plants,which is involved in the regulation of a variety of plant life processes.Previous studies about WRKY proteins have mostly focused on disease resistance and salt stress resistance.In recent years,the mechanisms of WRKY in flowering time control have been increasingly reported.In previous studies,two members of the WRK Y family,WRKY71 and WRK Y75,were found to regulate flowering time,but the molecular mechanism of their effect on flowering time has not been deeply studied.In this study,BjuWRK Y71 and BjuWRK Y75genes of B.juncea were cloned,and the interaction mechanisms of BjuWRKY71 and BjuWRKY75 with flowering integrators of BjuSOC1 and BjuFUL and flowering regulatory protein BjuHDA9 were studied via yeast two-hybrid,BiFC,yeast one-hybrid,double luciferase detection and other methods,so as to explore the molecular mechanism of WRK Y proteins in regulating flowering of B.juncea.The results are as follows:1.Cloning and sequence analysis of BjuWRKY71 and BjuWRKY75 in Brassica junceaTotal RNA from mustard leaves was isolated and reversely transcribed and then was taken as templates,BjuWRKY71 and BjuWRK Y75 genes were cloned by PCR.BjuWRK Y71 of 831bpCDS sequence and BjuWRKY75 of 438bp sequence were amplified,which encode 277 and 146 amino acids,respectively.Sequence alignment and phylogenetic tree analysis showed that BjuWRKY71 in Brassica juncea was closely related to WRKY71 in Brassica oleracea L,while BjuWRKY75 was closely related to WRKY75 in Brassica rapa.2.Expression patterns of BjuWRKY71 and BjuWRKY75.The expression of BjuWRK Y71 and BjuWRKY75 in different tissues was detected by qRT-PCR.The expression level of BjuWRK Y71 was the highest in flowers,followed by leaves,and the lowest in stems.The expression level of BjuWRKY75 was the highest in flowers and the lowest in leaves and stems.The expressions of BjuWRKY71 and BjuWRK Y75 were different at different stages.The expression of BjuWRK Y71 increased during the growth process,and the expression of BjuWRKY71 was up-regulated at15-25 days.The expression level of BjuWRK Y75 was higher at 25 days,but there was no significant difference between different periods.Subcellular location indicated that BjuWRKY71 and BjuWRKY75 are located in the nucleus.3.BjuWRKY71 and BjuWRKY75 promoted flowering of Brassica juncea.The fusion expression vectors pCAMBIA2301G-WRKY71 and pCAMBIA2301G-WRKY75,were respectively constructed and then transformed by Agrobacterium tumefaciens-mediated method.The transgenic plants overexpressing BjuWRK Y71 and BjuWRK Y75 were obtained in this study,respectively.Transgenic plants overexpressing BjuWRKY71 and BjuWRKY75 were identified by PCR.By observing the flowering phenotype of transgenic T1 plants,it was found that the flowering time of overexpressing BjuWRKY71 was 8 days earlier than that of J9W04,and that of overexpressing BjuWRK Y75 was 13 days earlier,indicating that BjuWRK Y71and BjuWRKY75 promoted flowering in B.juncea.4.The protein-protein interactions of BjuWRKY71 and BjuWRKY75 with BjuSOC1,BjuHDA9 and BjuFULIn order to investigate whether BjuWRKY71 and BjuWRKY75 affect flowering by directly interacting with flowering integron proteins BjuSOC1 and BjuFUL and flowering regulatory protein BjuHDA9,yeast double hybridization was used to detect the interactions between BjuWRKY71 and BjuWRKY75 proteins and them.The results showed that the fusion yeast strains of BjuWRKY71 and BjuWRKY75 with BjuSOC1,BjuHDA9 and BjuFUL could not grow colonies in Q DO/X/A（SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA）,indicating that BjuWRKY71 and BjuWRKY75 proteins did not directly interact with BjuSOC1,BjuHDA9 and BjuFUL proteins.The consistent results were confirmed by BiFC in this study.5.The protein-DN A interactions of BjuWRKY71 and BjuWRK Y75 with promoters of BjuSOC1 and BjuFULYeast one-hybrid assays showed that yeast strains of Y1H（pr SOC1+WRKY71）could grow white clones on SD/-Leu/AbA¹⁰⁰,while Y1H（pr SOC1+WRKY75）could not grow.It indicated that that BjuWRKY71 but not BjuWRKY75 was able to interact with SOC1 promoter.Yeast strains of Y1H（pr FUL+WRKY71）but not Y1H（pr FUL+WRKY75）could grow white clones on SD/-Leu/AbA⁴⁰⁰ medium,suggesting that only BjuWRKY71 interacted with BjuFUL promoter directly.Double luciferase results showed that the double fluorescence ratio of BjuWRKY71 to BjuSOC1 promoter was significantly higher than that of BjuSOC1promoter to pGreen Ⅱ 62-SK.However,compared with the the ratio of BjuWRK Y75 to BjuSOC1 promoter,there was no different in the double fluorescence ratio of BjuWRKY75 to BjuSOC1 promoter.It demonstrated that that BjuSOC1 promoter directly interacted with BjuWRKY71 but not with BjuWRKY75 in vivo.Similarly,the double fluorescence ratio of BjuWRKY71 to BjuFUL promoter was significantly higher than that of that of BjuFUL promoter to pGreen Ⅱ 62-SK,suggesting that BjuWRKY71 interacted with BjuFUL promoter.However,BjuWRKY75 could not bind to the promoter of BjuFUL,based on the double luciferase detection assays.The results were consistent with those of yeast one-hybrid,indicating that BjuWRKY71may regulate flowering through directly binding to the promoters of flowering integrators in B.juncea.In this study,we studied the molecular mechanism of BjuWRKY71 and BjuWRKY75 regulating flowering of B.juncea from the aspects of bioanalysis,gene expression analysis,transgenic function verification,protein-protein interaction,protein-DNA interaction and so on.Based on the above results,we found that BjuWRK Y71 and BjuWRK Y75 could promote flowering of B.juncea.They do not interact with the flowering integrons BjuSOC1 and BjuFUL and flowering regulatory protein BjuHDA9,while BjuWRKY71 interacts with the promoters of BjuSOC1 and BjuFUL,which may affect flowering by regulating the expression of BjuSOC1 and BjuFUL.There is no interaction between BjuWRKY75 and the BjuSOC1 promoter and the BjuFUL promoter,and its regulatory mechanism needs to be further studied.