Protein Interactions Of Flowering Integrator SOC1 With Inhibitors SVP And FLC In Brassica Juncea

In plants, flowering attaches great importance to developmental stage in which the transition from vegetative growth to reproductive growth affects the yield and quality of product organ. Flowering Integrator SUPPRESSOR OF CO OVEREXPRESSION1 (SOC1), Inhibitors FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP), which all code type Ⅱ MADS-box MIKC-type proteins, are critical to the regulation of plant development and flowering time. Although SOC1, SVP and FLC can regulate flowering time, the molecular mechanisms that they regulate flowering time are not clear. Moreover, the mechanisms of the interactions of SOC1 with SVP or FLC in Brassica juncea have not yet been reported.To further study the mechanisms, we have cloned SOC1 gene from’Qingye mustard’ germplasm of Brassica juncea and constructed Bimolecular fluorescence complementation (BiFC) recombinant expression vectors and yeast two hybrid recombinant expression vectors of SOC1, FLC, SVP and their mutants, then detected their interaction strengths by β-Galactosidase Assay. Also we have screened and identified the amino acids sites related to interactios of SOC1 with SVP and FLC protein, which will lay the foundation for further studying the mechanism of SOC1 in flowering time regulatory network.The main results were showed as follows:1. Subcloning and bioinformatics analysis of SOC1 gene in Brassica junceaTaking the first chain cDNA of stigma total RNA of stem apex as template from’Qingye mustard’, we got full length of SOCl gene, whose cDNA sequence was 639 bp encoding 213 amino acid residues, The protein that SOC1 encodes belongs to MIKC-type protein, having the closest genetic relationship with SOC 1 of Brassica carinata and Brassica napus. Meanwhile, we have predicted that the molecular weight is 24.37 kDa and pI is 9.18. According to the prediction and analysis of the secondary structure of SOC1 in Brassica juncea, we know that α-helix reaches to 56 percents of amino acid residues, (3-fold takes up 10% and coiled-coil is up to 34%.2. Detection of interaction between SOC1 and SVP in Brassica junceaConstructed pGBKT7-SOC1 (excluding signal peptide) yeast expression vector and transformed the recombinant yeast plasmid into competent cell by lithium acetate method, we got Y2HGold (pGBKT7-SOC1) that has no activation and toxicity by toxicity and activation detection. Fused Y2HGold(pGBKT7-SOC1) and Y187(pGADT7-SVP) which was provided by our lab, we knew diploid yeast Y187(pGADT7-SVP) x Y2HGold (pGBKT7-SOC1) can grow on SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A) with blue color.Meanwhile, constructed p2YC-SOCl and p2YN-SVP fluorescence expression vector, respectively and transformed the recombinant plasmid into Agrobacterium GV3101 by Cold and hot stimulus, we mixed equal amounts of these two kind of bacteria and co-infiltrated 4 to 6 true leaves of N. benthamiana. Observing at 488 nm excitation light by laser confocal scanning microscopy after 48h, we found that the epidermal cells of N. benthamiana which co-infiltrated p2YC-SOC1 and p2YN-SVP have fluorescence with yellow light. In summary, the interaction between flowering integrator SOC1 and inhibitor SVP exists in Brassica juncea.3. Detection of interaction between SOC1 and FLC in Brassica junceaFused Y2HGold (pGBKT7-SOC1) and Y187 (pGADT7-FLC) which was provided by the lab, we knew that diploid yeast Y187 (pGADT7-FLC) x Y2HGold (pGBKT7-SOC1) can grow on SD/-Leu/-Trp/AbA (DDO/A) and SD/-Ade/-His-Leu/-Trp (QDO) with white color, but can not grow on QDO/X/A.After constructing p2YN-FLC fluorescence expression vector, we transformed the recombinant plasmid into Agrobacterium GV3101 by cold and hot stimulus. By mixing equal amounts of the bacteria containing p2YC-SOC1 and co-infiltrating subsequently 4 to 6 true leaves of N. benthamiana, after 48h did we know that the epidermal cells of N. benthamiana co-infiltrated with p2YC-SOC1 and p2YN-FLC had no fluorescence at 488 nm excitation light by laser confocal scanning microscopy. In summary, there is no interaction between flowering Integrator SOC1 and inhibitor FLC in Brassica juncea.4. Detection of interaction among SOC1 mutants with SVP in Brassica junceaWe have constructed 5 SOC1 mutants in the K domain of SOC1 protein (SOC1V77K, SOC1P81K, SOC1K108V, SOC1R109Land SOC1C137K) and constructed pGBKT7-SOC1 mutants yeast expression vector, respectively. Transformed the recombinant yeast plasmid into competent cell by lithium acetate method, we got Y2HGold (pGBKT7-SOC1V77K), Y2HGold (pGBKT7-SOC1p81K), Y2HGold (pGBKT7-SOClK108V), Y2HGold (pGBKT7-SOC1R109L) and Y2HGold (pGBKT7- SOC1C137K). All of them have no activation and toxicity after toxicity and activation detection. Fused these 5 yeast transformants with Y187 (pGADT7-SVP), respectively, we knew that only diploid yeasts Y187 (pGADT7-SVP)×Y2HGold (pGBKT7-SOC1V77K) and Y187 (pGADT7-SVP) × Y2HGold (pGBKT7-SOC1P81K) can grow on QDO/X/A with blue color, while others can not.Meanwhile, we constructed p2YC-SOC1 mutants fluorescence expression vectors (p2YC-SOC1V77K, p2YC-SOC1P81K, p2YC-SOC1K108V, p2YC-SOC1R109L and p2YC-SOC1C137K), respectively, and transformed the recombinant plasmids into Agrobacterium GV3101 by cold and hot stimulus. Having identified the positive clones and mixed equal amounts of each kind of the bacteria with the bacteria containing p2YN-SVP, we co-infiltrated 4 to 6 true leaves of N. benthamiana. Observing at 488 nm excitation light by laser confocal scanning microscopy after 48h, we found that only the epidermal cells of N. benthamiana which co-infiltrated with p2YC-SOC1V77K and P2YN-SVP, p2YC-SOC1P81K and p2YN-SVP had fluorescence.5. Detection of interaction among SVP mutants with SOC1 in Brassica junceaBy constructing 4 SVP mutants in the K domain of SOC1 protein (SOC1V77K, SOC1P81C, SOC1K108V, SOC1R109L and SOC1C137K) and pGADT7-SVP mutants yeast expression vector, respectively, we transformed the recombinant yeast plasmid into competent cell by lithium acetate method and then got Y187 (pGADT7-SVPE90L), Y187 (pGADT7-SVPK104C), Y187 (pGADT7-SVPH1061) and Y187 (pGADT7-SVPR137L). Fusing these 4 yeast transformants with Y2HGold (pGBKT7-SOC1), respectively, we knew that diploid yeasts Y187 (pGADT7-SVPE90L) × Y2HGold (pGBKT7-SOC1), Y187 (pGADT7-SVPK104C) × Y2HGold (PGBKT7-SOC1) and Y187 (pGADT7-SVP H1061) × Y2HGold (pGBKT7-SOC1) can grow on QDO/X/A with blue color, whreas Y187 (pGADT7-SVPR137L) × Y2HGold (pGBKT7-SOC1) can not.Meanwhile, we constructed p2YN-SVP mutants fluorescence expression vector (P2YN-SVPE90L、P2YN-SVPK104C、p2YN-SVPH1061 and P2YN-SVPR137L), respectively, and transformed the recombinant plasmids into Agrobacterium GV3101 by Cold and hot stimulus. Bimolecular fluorescence complementation experiments showed that the epidermal cells of N. benthamiana which were co-infiltrated with p2YC-SOC1 and p2YN-SVPE90L,p2YC-SOC1 and p2YN-SVPK104C, p2YC-SOC1 and p2YN-SVPH1061 have fluorescence, but others can not.6. Analysis of the interaction strengthThe results of β-galactosidase activity detection and analysis of variance test showed that the protein interaction strength of both SVP with SOC1V77K and SVP with SOC1P81K are significantly higher than SVP with SOC1, meanwhile, strength of SVP with SOC1P81K is significantly higher than SVP with SOC1V77K. However, once the 108th,109th and 137th amino acid were mutated (SOC1K108V, SOC1R109L, SOC1C137K mutant), the interaction of SOCl and SVP will disappear. These indicate that these five amino acids of SOC1 are capable of regulating the aggregation strength of SOC1/SVP.The protein interaction strength of mutants SVPE90L, SVPK104C and SVPH1061 with SOC1 were significantly higher than SVP with SOC1. The difference of interaction strength of SVPK104C with SOC1 and SVPH1061 with SOC1 is not significant, but they were significantly higher than the mutant SVPE9OL with SOC1. However, mutation of the 137th amino acid (SVPR137L mutant) leads the interaction of SOC1 and SVP to disappear. This indicates that these four amino acids of SVP are capable of regulating the aggregation strength of SVP/SOC1.In addition, the interaction strength of hybrid combinations SOC1V77K × SVP, SOC1P81K× SVP are significantly higher than SOC1×SVPE90L, SOC1 × SVPK104C, SOC1×SVPH1061, that indicates the degree of influence on polymerization between SOC1 and SVP mutating at the 77th, 81th amino acids of SOC1 were higher than at the 90th,104th and 106th amino acids. The effects of using the mutations of K domain of SOC1 protein to regulate SOC1/SVP aggregation were likely to be more significantly than SVP.