Protein Interactions Of AGL15, AGL6 With SOC1, AGL24 Or SVP In Brassica Juncea

Flowering is an important and complex process for plant, flowering plants can feel the change of environment factors, and make the appropriate developmental regulation in order to get more possible chance to survive and reproduce maturation by their own genetic. This make the flowering-time as an important adaptive value for plants. A delay in floral induction may lead to a robust plant, but be late for seed maturation, in contrast, time to flower early can not guarantee that plants have sufficient energy reserves to produce offspring, so flowering time impact the next generation by control seeds maturation. Several genetic pathways control flowering time in response to environmental or endogenous conditions. Genetic pathways have some flowering integrators, include LEAFY, FT, SOC1, CO that promote flowering time and FLC, SVP, FLM which inhibit flowering time. Most of them are belong to MADS-Like protein, such as SOC1, AGL24 and SVP, they direct or indirect regulation of flowering-time, besides they functions of protein-protein and regulatory mechanism have been widely reported. The flowering Integrator SOC1, AGL24, SVP and FLC in Brassica juncea also have some reports. But the relationship between AGL6, AGL15 and SOC1, AGL24, SVP, FLC have not yet reported. So we cloned AGL6 and AGL15 gene from ‘QJ’ germplasm of Brassica juncea and research on the relationship between the AGL6, AGL15 and other flowering Integrator protein(SOC1, AGL24, SVP) by yeast two hybrid and Bimolecular fluorescence complementation(Bi FC), in order to further understand, we also study the relationship between the AGL15 promoter and other protein.The main results were showed as follows:1. Cloning and bioinformatics analysis of AGL6 and AGL15 gene in Brassica juncea Taking the first chain c DNA of stigma total RNA of stem apex as template from ‘Qingye mustard’, We got full length c DNA of AGL6 and AGL15 gene, AGL6 c DNA sequence was759 bp encoding 252 amino acid residues, AGL15 c DNA sequence was 795 bp encoding 264 amino acid residues. Taking genomic DNA from the leaves of Brassica juncea, we got full length DNA of AGL6 and AGL15 gene, AGL6 DNA sequence was 1771 bp, AGL15 DNA sequence was 1616 bp. Both the two gene contains eight exons and seven introns. The two protein belong to MIKC protein. We have predicted that the molecular weight of AGL6 is 28.85 k Da and p I is 9.15, and the AGL15 is 30.13 k Da and p I is 9.15. Both of them are unstable protein and having the closest genetic relationship with of AGL6/AGL15 Brassica napus. According to the prediction and analysis of the secondary structure of AGL6/AGL15 in Brassica juncea, we know that α-helix reaches to 40/70 percents that mainly in K domain.2. Cloning and bioinformatics analysis of the AGL15 promoter Taking genomic DNA from the leaves of Brassica juncea, using Genome Walking method, we successfully obtained 890 bp AGL15 promoter sequence. Phylogenetic analysis of the AGL15 promoter showed it had the closed relationship with Brassica juncea AGL15. Bioinformatics analysis of the AGL15 promoter sequence found that it contains a core promoter element of TATA-box, CGTCA-motif, TGGTTT-ARE promoter element.3. Detection of interaction between AGL6 and SOC1, AGL24, SVP in Brassica juncea Constructed p GADT7-AGL6 and p GBKT7-AGL6(excluding signal peptide) yeast expression vector and transformed the p GBKT7-AGL6 into competent cell Y2 HGold by lithium acetate method, we got Y2HGold(p GBKT7-AGL6) that has no activation and toxicity by toxicity and activation detection. We knew diploid yeast Y2HGold(p GBKT7-AGL6)×Y187(p GADT7-SOC1) 、(p GBKT7-AGL6)×Y187(p GADT7-AGL24) 、Y2HGold(p GBKT7-AGL6)×Y187(p GADT7-SVP) can grow on SD/–Ade/–His/–Leu/–Trp/X-a-Gal/Ab A(QDO/X/A)with blue color. We constructed p GPTVⅡ.YFPN-AGL6 and p GPTVⅡ.YFPC-AGL6 fluorescence expression vector by connecting AGL6 and the N-terminal vctor p GPTVⅡ.YFPN and p GPTV Ⅱ.YFPCof Bi FC, we co-infiltrated 4 to 6 true leaves of N.benthamiana. Observing at 488 nm excitation light by fluorescence microscope after 48 h, we found that the cells of N. benthamiana which co-infiltrated GV3101[p GPTVⅡ.YFPN-AGL6] ×GV-3101[p-GPTV Ⅱ.YFPC-SOC1], GV3101[p GPTV Ⅱ.YFPN-AGL6] ×GV3101[p GPTVⅡ.YFPC-AGL24], GV3101[p GPTVⅡ.YFPN-AGL6] ×GV3101[p GPTVⅡ.YFPC-SVP] have fluorescence with yellow light. In summary, the interaction between flowering integrator AGL6 and SOC1, AGL24, SVP exists in Brassica juncea.4. Detection of interaction among AGL6 amino acid deletion mutants with SOC1, AGL24,SVP in Brassica juncea We predict the protein- protein binding sites of AGL6 by ISS, and we constructed three amino acid deletion mutants of AGL6: AGL6D-NN(delete the 79 th and 80 th two asparagine ammonia(N)), AGL6D-RT(delete the 105 th arginine(R) and 106 th threonine(T)) and AGL6D-NNRT(delete four amino acids mentioned earlier). We constructed p GBKT7-AGL6D-NN, p GBKT7-AGL6D-RT, p GBKT7-AGL6D-NNRT yeast expression vector. After toxicity and activation detection, fused these three Y2 HGold yeast with Y187(p GADT7-SOC1), Y187(p GADT7-AGL24) and Y187(p GADT7-SVP) respectively, we find that except Y2HGold(p GBKT7-AGL6 D-NNRT) ×Y187(p GADT7-SVP) cannot grow on QDO/X/A, others can grow on QDO/X/A tablet and have blue color. Meanwhile, constructed GPTV Ⅱ.YFPN-AGL6 D-NN, p GPTV Ⅱ.YFPN-AGL6D-RT and p GPTVⅡ.YFPN-AGL6D-NNRT amino acid deletion mutants fluorescence expression vector. We mixed equal amounts of these two kind of bacteria, one is GV3101[p GPTVⅡ.YFPC-SVP], another is GV3101[p GPTV Ⅱ.YFPN-AGL6D-NN]/GV3101[p GPTVⅡ.YFPN-AGL6D-RT]/GV3101[p GPTVⅡ.YFPN-AGL6D-NNRT] and co-infiltrated 4 to 6 true leaves of N. benthamiana. Observing at 488 nm excitation light by fluorescence microscope after 48 h, it shows only the GV3101[p GPTV Ⅱ.YFPN-AGL6D-NN] ×GV3101[p GPTV Ⅱ.YFPC-SVP] and GV3101[p GPTV Ⅱ.YFPN-AGL6D-RT] ×GV3101[p GPTV Ⅱ.YFPC-SVP] have fluorescence with yellow light. But the GV3101[p GPTV Ⅱ.YFPN-AGL6D-NNRT] ×GV3101[p GPTV Ⅱ.YFPC-SVP] have no fluorescence with yellow light. It means that AGL6D-NN and AGL6D-RT can interacted with SVP protein, but AGL6D-NNRT cannot. It maybe that the four amino acid can regulate the interaction of AGL6 and SVP.5. Detection of interaction between AGL15 and SOC1, AGL24, SVP, AGL6 in Brassica juncea Constructed p GADT7-AGL15 and p GBKT7-AGL15(excluding signal peptide) yeast expression vector and transformed the p GBKT7-AGL15 into competent cell Y2H-Gold by lithium acetate method, we got Y2HGold(p GBKT7-AGL15) that has no activation and toxicity by toxicity and activation detection. Fused Y2HGold(p GBKT7-AGL15) with Y187(p GADT7-SOC1), Y187(p GADT7-AGL24) and Y187(p GADT7-SVP). We knew only the diploid yeast(p GBKT7-AGL6)×Y187(p GADT7-AGL24) can grow on QDO/X/A with blue color, and the diploid yeast Y2HGold(p GBKT7-AGL6)×Y187(p GADT7-SVP),Y2HGold(p GBKT7-AGL6)×Y187(p GADT7-SOC1)cannot grow on QDO/X/A. We constructed p GPTV Ⅱ.YFPN-AGL15 fluorescence expression vector by connecting AGL15 and the N-terminal vctor p GPTVⅡ.YFPN of Bi FC. Observing at 488 nm excitation light by fluorescence microscope after 48 h, we found that the cells of N. benthamiana which co-infiltrated GV3101[p GPTVⅡ.YFPN-AGL6] ×GV3101[p GPTVⅡ.YFPC-AGL24] have fluorescence with yellow light, other is not. In summary, the interaction between flowering integrator AGL15 and SOC1, SVP is not exists in Brassica juncea except for AGL24. 6. Detection of interaction among AGL15 amino acid deletion mutants with AGL24 in Brassica juncea We predict the protein- protein binding sites of AGL15 by ISS too, and we constructed three amino acid deletion mutants of AGL15: AGL15D-EK(delete the 107 th glutamic(E) acid and 108 th lysine(K)), AGL15D-R(delete the 170 th arginine(R)) and AGL15D-EKR(delete four amino acids mentioned earlier). We constructed p GBKT7-AGL15D-EK, p GBKT7-AGL15D-R, p GBKT7-AGL15D-EKR yeast expression vector and transformed the it into competent cell Y2 HGold by lithium acetate method then got Y2HGold(p GBKT7-AGL15D-EK), Y2HGold(p GBKT7-AGL15D-R) and Y2HGold(p GBKT7-AGL15D-EKR). After toxicity and activation detection, fused these three Y2 HGold yeast with Y187(p GADT7-AGL24) respectively, we find that only Y2HGold(p GBKT7-AGL15D-EK) ×Y187(p GADT7-AGL24) can grow on QDO/X/A with blue color, others cannot grow on QDO/X/A tablet. Meanwhile, constructed GPTVⅡ.YFPN-AGL15D-EK, p GPTVⅡ.YFPN-AGL15D-R and p GPTVⅡ.YFPN-AGL15D-EKR amino acid deletion mutants fluorescence expression vector. Observing at 488 nm excitation light by fluorescence microscope after 48 h, it shows only the GV3101[p GPTV Ⅱ.YFPN-AGL15 D-EK] ×GV3101[p GPTV Ⅱ.YFPCAGL24] have fluorescence with yellow light. But the GV3101[p GPTVⅡ.YFPN- AGL-15D-R] ×GV3101[p GPTVⅡ.YFPC-AGL 24] and GV3101[p GPTVⅡ.YFPN-AGL15D-EKR] ×GV3101[p GPTVⅡ.YFPC-AGL 24] have no fluorescence with yellow light. It means that AGL15D-R and AGL15D-EKR can not interacted with AGL24 protein, but AGL15 D-EK can. It maybe means that the 170 th arginine can regulate the interaction of AGL15 and AGL24.7. Detection of the interactions between AGL15 promoter and SOC1, AGL24, AGL15 or AGL6 proteins We constructed the yeast recombinant plasmids p Ab Ai-AGL15 Q and we integrated the linearized p Ab Ai-AGL15 Q plasmid into Y1 HGold yeast and Y1H(p Ab Ai-AGL15Q) and do Ab A concentration screening experiment. After that, we transformed recombinant plasmid p GADT7-SOC1, p GADT7-AGL24, p GADT7-AGL15 and p GADT7-AGL6 into Y1H(p Ab Ai-AGL15Q) by lithium acetate method. Than we found Y1HGold(p Ab AiAGL15Q)-SOC1, Y1HGold(p Ab Ai-AGL15Q)-AGL24 and Y1HGold(p Ab AiAGL15Q)-AGL15 can gown normally on SD/-Leu/Ab A-150, but Y1HGold(p Ab AiAGL15Q)-AGL6 cannot. We use AGL15 promoter constructed dual luciferase technology recombinant plasmid p Green Ⅱ 0800-LUC-AGL15 Q vector, and constructed protein recombinant plasmid p GreenⅡ62-SK-AGL15 and p GreenⅡ62-SK-AGL6, the other two recombinant plasmids was provided by our lab, and transformed the recombinant plasmids into Agrobacterium GV3101 by cold and hot stimulus, then we got GV3101[p GreenⅡ0800-LUC-AGL15Q], GV3101 [p GreenⅡ62-SK-AGL15], GV3101 [p GreenⅡ62-SK-AGL6], GV3101 [p GreenⅡ62-SK-SOC1], GV3101 [p GreenⅡ62-SK-AGL24], We mixed equal amounts of these two kind of bacteria, one is AGL 15 promoter, another is protein(SOC1, AGL24, AGL15 or AGL6), take material after 48 hours later, and use the Glo Max-Multiinstrument to read numeric, then analysis the data. We find SOC1, AGL24 or AGL15 protein with AGL15 promoter show higher value than control, but AGL6 protein is not. It suggested that the AGL15 promoter in Brassica juncea could interact with SOC1, AGL24 or AGL15 proteins.