| Brassica juncea,a kind of vegetable crop in Brassica,is widely cultivated in South of China.The yield and quality of the product organs will be seriously affected by the flowering time.Previous studies showed that a large number of genes the that regulating the flowering time have been found in Arabidopsis,which formed a huge and subtle gene regulation network.MADS-box transcription factors(such as flowering inhibiting factor AGL18)play crucial roles in the network.However,the relationships between AGL18 and other flowering time genes in the regulation network remain unclear,and the molecular mechanisms of Brassica juncea AGL18 in regulating flowering time have not been thoroughly revealed.To clarify the regulation mechanisms of AGL18 in flowering pathways,we cloned AGL18 s gene from Brassica juncea,and detected the protein-protein interactions of AGL18s/SOC1 and AGL18s/AGL24 using yeast two-hybrid and Bi FC,the protein-DNA interactions of AGL18s/SOC1 and AGL18s/AGL24 using yeast one-hybrid and Dual-Glo? Luciferase Assay System.Then the interaction regions of AGL18 s with SOC1/SOC1 and/or AGL24/AGL24 were selected,respectively.Furthermore,we also screened and verified the crucial amino acids that participated in the interactions of AGL18s/AGL24.Meanwhile,in order to verify and analysis the function of AGL18 in vivo,the recombinant plasmids p BI35S::s AGL18?1-3 and p BI35S::a AGL18?1-3 were constructed and transformed into Agrobacterium tumefaciens cells to infiltrate into the Brassica juncea and tobacco.This work provided valuable information for in-depth studies on the molecular mechanisms of AGL18 in regulating the flowering time in Brassica juncea.The main results were showed as follows:1.Molecular cloning and bioinformatics analysis of AGL18 gene in Brassica junceaIn this study,AGL18-1 gene from reproductive shoots as well as AGL18-2 and AGL18-3from vegetative shoots in‘QJ' germplasm of Brassica juncea,were cloned respectively.AGL18-1,AGL18-2 and AGL18-3 were members of AGL18 family and respectively encoded 257,257 and 258 amino acids and belong to MIKCc-type proteins.Sequence analysis showed that the nucleotide sequences of the AGL18 s were similar to Brassica rapa and Brassica napus.The predicted molecular weight was 29.01 k Da for AGL18-1,28.73 k Da for AGL18-2 and 29.34 k Da for AGL18-3,and the p I was 5.65 for AGL18-1,5.15 for AGL18-2 and 5.86 for AGL18-3.Meanwhile,three of the Bj AGL18 family members were hydrophilic and unstable proteins.2.AGL18?1-3 could not interact with SOC1 protein in Brassica junceaIn this study,pGADT7-AGL18?1-3 and pGBKT7-AGL18?1-3 were served as bait plasmids,respectively.Yeast two-hybrid assays showed that AGL18?1-3 proteins did not interact with SOC1 in Brassica juncea.However,Bi FC assays showed that the co-expression combination strains had not produced green fluorescent signals in the leaves,which were co-infiltrated by N.Benthamiana,as observed with confocal laser scanning microscopy(CLSM),suggesting that the AGL18 s could not interact with SOC1 in vivo.3.AGL18-1 interact with SOC1 promoter in Brassica junceaYeast one-hybrid showed that p Ab Ai-SOC1 fused only with p GADT7-AGL18-1 could grown normally on SD/-Leu/Ab A100 plates,suggesting that only the AGL18-1 protein interact with SOC1 promoter in Brassica juncea.Furthermore,Dual-luciferase activity assays showed that the ratio of the firefly luciferase and renilla luciferase of the experimental group was significantly higher than that of the negative control group,indicating that AGL18-1 interact with SOC1.4.The M domain of AGL18-1 mediate the interaction of AGL18-1/SOC1We have confirmed that AGL18-1 interact with SOC1 promoter.To screen the interaction region of AGL18-1/SOC1,the M and IKC domains of AGL18-1 were truncated,respectively.Yeast one-hybrid assays showed that only the M domain truncated could interact with SOC1 promoter.It indicated that the M domain of AGL18-1 was the key region that interact with SOC1 in Brassica juncea.5.AGL18?1-3 interact with AGL24 protein in Brassica junceaIn the study,p GBKT7-AGL18?1-3 were served as bait plasmids and p GADT7-AGL24 was served as prey plasmid.Yeast two-hybrid assays showed that AGL18?1-3 could interact with AGL24 and activate the transcriptional expression of the reporter genes HIS3,ADE2,MEL1 and AUR1-C.Bi FC assays showed that the tobacco leaves,which were co-infiltrated with N.Benthamiana,exhibited green fluorescence with CLSM.These results indicated that AGL18 s proteins could interact with AGL24 in vivo.6.The K domains mediating the interactions of AGL18s/AGL24 in Brassica junceaTo screen the specific interaction regions of AGL18s/AGL24,the M,K,MIK and KC domains of AGL18?1-3 were respectively truncated to construct the yeast expression vectors.Yeast two-hybrid assays showed that the K,MIK,KC domains of AGL18?1-3could interact with AGL24.The results suggested that the K domains played crucial roles in mediating the interactions of AGL18 s and AGL24.7.AGL18-1 interact with AGL24 in Brassica junceaYeast one-hybrid showed that p Ab Ai-AGL24 fused only with p GADT7-AGL18-1 could grown normally on SD/-Leu/Ab A100 plates.It suggested that only the AGL18-1 protein interact with AGL24 promoter in Brassica juncea.Furthermore,Dual-luciferase activity assays showed that the ratio of the firefly luciferase and renilla luciferase of the experimental group was significantly higher than that of the negative control group,indicating that AGL18-1 interact with AGL24.8.The M and K domains mediating the interaction of AGL18-1/AGL24 in Brassica junceaThe M,IKC,K,MIK and MK domains of AGL18?1-3 were respectively truncated to screen the specific interaction regions of AGL18s/AGL24.Yeast one-hybrid assays showed that all the truncateds can interact with AGL24.The results suggested that the M and K domains played crucial roles in mediating the interactions of AGL18 s and AGL24.9.Interactions of AGL24 with point mutants in K domains of AGL18?1-3 in Brassica junceaThe key amino acid sites of AGL18?1-3 that involved in protein-protein interaction were predicted online by ISIS,and then eleven mutants in K domains of AGL18?1-3were consructed successfully.Yeast two-hybrid,Bi FC and ?-Galactosidase assays of the mutations in their K domains showed that the AGL18-2L113 F,AGL18-2E116 H,AGL18-2L118 F,AGL18-2K165 T,AGL18-3L114 P,AGL18-3E117 G,AGL18-3R118 G and AGL18-3L119 Pmutants still interacted with AGL24 in yeast and in vivo.In addition,these mutations could enhance or weaken but not eliminate the interactions between AGL18-2/AGL24 and/or AGL18-3/AGL24,indicating that they are not critical sites in regulating the interactions of AGL18-2/AGL24 and AGL18-3/AGL24.The interaction strength of AGL18-1/AGL24 was heavily weakened by AGL18-1L114 V and AGL18-1E117 V,though interaction could still occur.However,the AGL18-1K190 I mutant could not interact with AGL24,indicating that the 190 thamino acid of AGL18-1 was crucial in mediating the formation of AGL18-1/AGL24 dimers.10.Construction of the sense and anti-sense expression vectors and genetic transformation of AGL18?1-3To explore the function of AGL18?1-3 genes in Brassica juncea,we respectively inserted the full-length sequences of AGL18?1-3 into the plant binary vector p BI121 forwardly and reversely to construct recombinant plasmids p BI35S::s AGL18?1-3 and p BI35S::a AGL18?1-3,and then transformed into Agrobacterium tumefaciens cells(GV3101)for expanded culture to infiltrate into the hypocotyl of Brassica juncea and the leaves of tobacco.And we obtained more than 100 transgenic tobacco and about 30 transgenic Brassica juncea,and observed the phenotypic changes in the transgenic plants at the same time. |
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