| Brassica juncea Coss.(Mustard) is an important cruciferae vegetable cultivated widely in South China. The appropriate timing of flowering will affect the yield and quality of mustard product organ. After decades of research, a large number of genes related to flowering time have been found in Arabidopsis thaliana. However, the molecular mechanisms of regulating flowering time in Brassica juncea are not thoroughly revealed. So far, the mechanisms of the interactions of AGL24 protein with SOC1 gene in Brassica juncea have not been reported.In order to clarify the mechanisms, we detected the interactions of SOC1 full-length promoter, truncated promoter or promoter mutant with AGL24 protein using both yeast one-hybrid system and dual-luciferase report gene system. And then we screened the critical region related to interactions of SOC1 promoter with AGL24 protein. Meanwhile, in order to study the interactions in vivo, recombinant plasmid pBI-AGL24 was constructed and then transformed into mustard by the method of Agrobacterium mediation. The results of our research provided valuable information for further studying the expression and regulation of SOC1.The main results were showed as follows: 1. Cloning and bioinformatics analysis of SOC1 gene in Brassica junceaTaking the first chain c DNA of stem apex as template from Brassica juncea, we got full length of AGL24 gene with 666 bp, enconding 221 amino acid residues. At the same time, we have predicted that the AGL24 protein was composed of 20 kinds of amino acids, among which Leu and Ser had the highest proportion, the relative molecular weight was 24.902 kDa, and the p I was 7.67. 2. Detection of the interactions between SOC1 full-length promoter and AGL24 proteins in Brassica junceaWe drew the recombinant strain to coated on SD/-Ura/Ab A plates to screen a minimal inhibitory concentration of AbA for recombinant strain Y1H(p AbAi-SOC1). The results showed that the recombinant strain couldn’t grow on the plates with the concentration 100 ng/m L AbA. Therefore, we inferred that 100 ng/mL AbA was the minimal inhibitory concentration for the growth of recombinant strain Y1H(pAbAi-SOC1). The yeast recombinant plasmid pGADT7-AGL24 was transformed into the yeast competent cells with Y1H(p AbAi-SOC1) and formed diploid yeast strain Y1H(SOC1+AGL24), which grew normally in the SD/-Leu/AbA100 plates. It suggested that the SOC1 promoter of mustard could interact with AGL24 protein.Meanwhile, dual-luciferase expression vectors LUC-SOC1 and SK-AGL24 were respectively constructed and transformed into Agrobacterium GV3101(pSoup). The above Agrobacterium solutions respectively containing LUC-SOC1 and SK-AGL24 were mixed at the ratio of 1:9 and then co-infiltrated into 6 true leaves of N. benthamiana and co-cultured for 60 h. Then the acitivities of firefly luciferase and renilla luciferase in the leaves of N. benthamiana were measured via Glo Max?-Multi+. In addition, the ratio of firefly luciferase to renilla luciferase was calculated. The results showed that the ratio of the experimental group was significantly higher than that of the negative control group, suggesting that the interaction between SOC1 promoter and AGL24 protein exists in Brassica juncea. 3. Detection of interactions between SOC1 truncated forms and AGL24 proteins in Brassica junceaTo figure out the critical interacting regions between SOC1 promoter and AGL24 protein. The minimal concentrations of AbA that respectively inhibited the growth of recombinant strain Y1H(pAbAi-SOC1-1), Y1H(pAbAi-SOC1-2) and Y1H(pAbAi-SOC1-3) were screened and set to be 100 ng/m L, 150 ng/mL and 100 ng/m L, respectively. Next, we prepared yeast competent cells with Y1H(pAbAi-SOC1-1), Y1H(pAbAi-SOC1-2) and Y1H(pAbAi-SOC1-3) for the co-transformation of yeast recombinant plasmid pGADT7-AGL24. We found that only the diploid yeast strains of Y1H(SOC1-2+AGL24) grew normally on the SD/-Leu/AbA media which contained the corresponding background concentrations of AbA, whereas others did not.In order to confirm our results, and to further test whether the SOC1 truncated forms interact with AGL24 protein. Dual-luciferase expression vectors LUC-SOC1-1, LUC-SOC1-2 and LUC-SOC1-3 were respectively constructed and transformed into Agrobacterium GV3101(pSoup) by Cold and hot stimulus. The above Agrobacterium solutions were mixed together with Agrobacterium solution containing SK-AGL24 at the ratio of 1:9 and then co-infiltrated into 6 true leaves of N. benthamiana and cultured for 60 h. The activities of firefly luciferase and renilla luciferase were measured in GloMax?-Multi+. And then the ratio of firefly luciferase to renilla luciferase was calculated. The results showed that the ratio of all experimental groups was significantly higher than that of the negative control group, suggesting that each of SOC1 truncate forms can interact with AGL24 protein in Brassica juncea. 4. Detection of the interactions between the CArG-box mutant of SOC1 promoter and AGL24 proteins in Brassica junceaIn order to strengthen our experimental results, and to figure out whether the DNA-binding site CArG-box on the SOC1 promoter have specificity or not,To figure out the critical interacting regions between SOC1 promoter and AGL24 protein, we extracted two kinds of yeast recombinant plasmid(one is A-T base exchanged in CArG-box and the other is CArG-box deleted). Then, the recombinant strains were coated on SD/-Ura/AbA plates to screen minimal inhibitory concentrations of AbA for the growth of the recombinant strains. The results showed that the minimal concentrations of AbA that inhibited the growth of recombinant strains Y1H(pAbAi-SOC1-1E), Y1H(pAbAi-SOC1-2E) and Y1H(pAbAi-SOC1-3E) were 100 ng/mL, 150 ng/m L and 200 ng/m L, respectively. However, the AbA concentrations of inhibiting the growth of recombinant strains Y1H(p AbAi-SOC1-1D), Y1H(pAbAi-SOC1-2D) and Y1H(pAbAi-SOC1-3D) were 100 ng/mL. Further more, the yeast competent cells with SOC1 promoter were co-transformed with yeast recombinant plasmid pGADT7-AGL24, and formed the diploid yeast strain, which could not grow on SD/-Leu/Ab A meida with the corresponding background concentrations of AbA.At the same time, dual-luciferase expression vectors LUC-SOC1-1E, LUC-SOC1-2E, LUC-SOC1-3E, LUC-SOC1-1D, LUC-SOC1-2D and LUC-SOC1-3D were respectively constructed and then transformed into Agrobacterium GV3101(pSoup) by Cold and hot stimulus. The above Agrobacterium solutions were respectively mixed with Agrobacterium solution containing SK-AGL24 at the ratio of 1:9 and then co-infiltrated into 6 true leaves of N. benthamiana and co-cultured for 60 h. Next, the activities of firefly luciferase and renilla luciferase were measured in GloMax?-Multi+. Finally, the ratio of firefly luciferase to renilla luciferase was calculted. The results showed there were no significant difference between the ratio of all experimental groups and that of the negative control group. It suggested that SOC1 promoter had three CArG-box moitfs, which probably regulated the DNA-protein interactions. 5. Expression vector construction of AGL24 gene and transformation into mustardTo construct transgene recombination plasmids, primers were designed according to AGL24 gene sequence and the multiple cloning sites of pBI121 vector. Next, we constructed expression plasmid pBI121-AGL24 and then infected mustard seedling hypocotyl on MS medium with proper plant hormone. After a series of experiments of pro-cultur, infection, co-culture, selective and differentiation culture, growing into seedlings and transplanting, we obtained transgenic mustard plants which overexpressed AGL24 gene. |
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