Protein Interactions Of Flowering Signal Integrator AGL24 With Regulation Factors SVP, FLC And SOC1 In Brassica Juncea And Screening The Critical Sites Of Protein Interactions

It is a complex and delicate process of regulation of flowering time in Brassica juncea, which needs to integrate environmental signals and endogenous signals. Under appropriate conditions, the transition from vegetative development to flowering can perform. The MADS-box transcription factors play a crucial role in these processes that occupy key positions in the complex regulatory networks in the flowering-time control. There are FLC and SVP that act to inhibit flowering among them, whereas SOC1 and AGL24(AGAMOUS-LIKE 24) can promote flowering. AGL24, SOC1, SVP, FLC exercise their functions in regulating the flowering network through proteins interactions which belong to MIKC-type proteins. However, the protein-protein interactions attach great importance to their functions and biological processes which they regulate. The majority of proteins having MADS domain are involved in protein-protein interactions that are conservative. Up to now, about the function and mechanism of SVP, SOC1, FLC have been widely reported, but the molecular mechanisms of AGL24 are rarely reported. In order to study whether AGL24 can interact with the same class of proteins in plants and filter out the key amino acid sites determining the protein interactions, we use both the yeast two-hybrid system and bimolecular fluorescence complementation technology. The study not only can further reveal underlying contact in the molecular regulatory network, but also further account for biological processes at the molecular level in vivo.1. Subcloning and bioinformatics analysis of AGL24 gene in Brassica junceaUsing the first chain cDNA of stigma total RNA of stem apex as template from’Qingye mustard’, we got full length of AGL24 gene, whose cDNA sequence is 666bp encoding the protein including 221 amino acid residues that belongs to MIKC-type protein. The AGL24 in Brassica Juncea has the closest genetic relationship with AGL24 of Brassica napus and Populus tomentosa is the relative farthest. AGL24 have the characteristic of hydrophily and secondary structure prediction analysis shows the a-helix is up to 50% that is mainly in K domain and β-fold and coiled-coil accounts for 12% and 38%, respectively. At the same time, our test have been predicted the three-dimensional image of AGL24 protein.2. Detection of interactions of AGL24 with SVP and FLC in Brassica juncea.We have constructed pGADT7-AGL24 and pGADT7-AGL24M which is a truncated form removed the MADS domain of AGL24 (excluding signal peptide) yeast expression vectors and then transformed the recombinant yeast plasmids into competent cell by lithium acetate method. We have got Y187 (pGADT7-AGL24) and Y187 (pGADT7-AGL24) and both of them have no activation and toxicity after toxicity and the activation detection. Fused Y187 (pGADT7-AGL24) and Y187 (pGADT7-AGL24M) with Y2HGold(pGBKT7-SVP) and Y2HGold(pGBKT7-FLC) respectively, we obtained diploid yeasts Y187(pGADT7-AGL24)× Y2HGold(pGBKT7-SVP), Y187(pGADT7-AGL24M)× Y2HGold(pGBKT7-SVP), Y187(pGADT7-AGL24) ×Y2HGold(pGBKT7-FLC), Y187(pGADT7-AGL24M)×Y2HGold(pGBKT7-FLC). None of them can grow on SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA(QDO/X/A).We have constructed the recombinant plasmids YCEG and YCEGM with connecting AGL24 and the truncated form removed the MADS domain to the C-terminal vector (pSPYCE-35S) of BiFC respectively. Meanwhile, connected SVP and FLC respectively to the N-terminal vector (pSPYNE-35S) of BiFC, we have got recombinant plasmids YNES and YNEF. Then transformed the recombinant plasmids into Agrobacterium GV3101 by Cold and hot stimulus, we have mixed equal amounts of these two kind of bacteria(YCEG X YNES, YCEG X YNEF, YCEGM ×YNES, YCEGM × YNEF) and co-infiltrated 4 to 6 true leaves of N. benthamiana. Observed at 488 nm excitation light by laser confocal scanning microscopy after 48 h, did we found no fluorescence perform at epidermal cells of N. benthamiana. In summary, the flowering signal integrator AGL24 and the truncated form does not interact with SVP or FLC in Brassica juncea.3. Detection of interaction between AGL24 and SOC1 in Brassica junceaAfter fusing Y187 (pGADT7-AGL24) and Y187 (pGADT7-AGL24M) with Y2HGold (pGBKT7-SOC1) and Y187 (pGBKT7-SOC1M) respectively, we found that both diploid yeast Y187 (pGADT7-AGL24)×Y2HGold (pGBKT7-SOC1) and diploid yeast Y187 (pGADT7-AGL24M)×Y2HGold (pGBKT7-SOClM) can grow on SD/-Leu/-Trp/AbA(DDO/A) and SD/-Ade/-His-Leu/-Trp(QDO) with white color, also can grow on QDO/X/A with blue color. However, through repeated tests did we know neither the diploid yeast Y187(pGADT7-AGL24)×Y2HGold(pGBKT7-SOC1M) nor the diploid yeast Y187(pGADT7-AGL24M)×Y2HGold (pGBKT7-SOC1) could grow on DDO/A, QDO or QDO/X/A.Using Yeast β-Galactosidase Assay Kit of Thermo company for measuring β-galactosidase activity to analyze protein-protein interactions strength of crosses pGADT7-AGL24 X pGBKT7-SOC1 and pGADT7-AGL24MxpGBKT7-SOC1, we found that the average activity (5.05 Miller Units) of interaction between full-length proteins AGL24 and SOC1 is significantly lower than the activity (8.24 Miller Units) between truncations AGL24M and SOC1M, which indicated the MADS domains of AGL24 and SOC1 have a certain inhibitory for their interaction.Connected SOC1 and the truncated form removed the MADS domain of SOC1 to the C-terminal vector (pSPYNE-35S) of BiFC respectively, we constructed the recombinant plasmids YNEO and YNEOM. Mixed the Agrobacterium containing YCEG or YCEGM with Agrobacterium containing YNEO or YNEOM at the same amount respectively and then co-infiltrated 4 to 6 true leaves of N. benthamiana, we found that the epidermal cells of N. benthamiana which were co-infiltrated with YCEG and YNEO after 48 h performance yellow fluorescence by laser confocal scanning microscopy at 488 nm excitation light. And it is the same with the cross YCEG X YNEO. However, the epidermal cells of N. benthamiana which was co-infiltrated with YCEGM and YNEO or YCEG and YNEOM have no fluorescence. In summary, the flowering signal integrators AGL24 can interact with SOC1 and both of them removed the MADS domains also have interactions. Either of them removed the MADS domain has not protein-protein interactions.4. Screening of the key amino acid sites of AGL24 involved in protein-protein interactions with SOC1We have predicted three key amino acid residue sites in K domain of AGL24 protein and constructed 3 AGL24 mutants (AGL24Q101L, AGL24R137L, AGL24E169L). Then constructed pGADT7-AGL24Q107L, pGADT7-AGL24R137L, pGADT7-AGL24E169L mutants yeast expression vector respectively and transformed the recombinant yeast plasmid into competent cell by lithium acetate method, we got Y187(pGADT7-AGL24Q107L), Y187(pGADT7-AGL24R137L) and Y187 (pGADT7-AGL24E169L). They have no activation and toxicity after toxicity and the activation detection. Fused these three yeast transformants with Y187 (pGADT7-SOC1) respectively, we knew that diploid yeasts Y187(pGADT7-AGL24R137L)×Y2HGold (pGBKT7-SOC1) and Y187(pGADT7-AGL24E169L)×Y2HGold(pGBKT7-SOC1) can grow on QDO/X/A with blue color, whereas only the diploid yeast Y187(pGADT7-AGL24Q107L)× Y2HGold(pGBKT7-SOC1) can’t grow on QDO/X/A.Yeast β-galactosidase activity analysis shows that compared the interaction strength of AGL24 with SOC1 (activity 4.44) with mutants at the 137th and 169th points of AGL24 with SOC 1 (activity 3.11 and 3.70), the difference is not significant which indicates that the two points are not the key amino acid sites regulating the interaction strength of AGL24 with SOC1.Connected three mutants of AGL24 to the C-terminal vector (pSPYNE-35S) of BiFC respectively, we constructed the recombinant plasmids YCEGQ107L, YCEGR137L, YCEGE169L, we use cold and hot stimulus to transform the recombinant plasmids into Agrobacterium GV3101. Mixed the Agrobacterium containing YCEGQ107L, YCEGR137L or YCEGE169L with Agrobacterium containing YNEO at the same amount respectively and then co-infiltrated 4 to 6 true leaves of N. benthamiana, we found that the epidermal cells of N. benthamiana which were co-infiltrated with YCEG and YNEO after 48 h performance yellow fluorescence by laser confocal scanning microscopy at 488 nm excitation light. It is the same with the cross YCEGE169L×YNEO. However, the epidermal cells of N. benthamiana co-infiltrated by the cross YCEGQ107L×YNEO has no fluorescence. All in all, mutations in the 137th and 169th amino acid residues have no obvious effects on the interactions of AGL24 with SOC1, but the mutation in the 107th amino acid residues have suppressed the interaction between AGL24 and SOC1, which shows the 107th amino acid residue is the key point participating the protein-protein interaction.5. Screening of the key amino acid sites of SOC1 involved in protein-protein interactions with AGL24Transforming the recombinant yeast plasmid pGBKT7-SOC1V77K、pGBKT7-SOCl1P81K、 pGBKT7-SOC1K108V、pGBKT7-SOC1R109L and pGBKT7-SOC1C137K into competent cell by lithium acetate method, we got Y2HGold(pGBKT7-SOC1V77K), Y2HGold(pGBKT7-SOC1P81K), Y2HGold(pGBKT7-SOC1K108V), Y2HGold(pGBKT7-SOC1R109L) and Y2HGold(pGBKT7-SOC1C137K). They have no activation and toxicity after toxicity and the activation detection. Fused these 5 yeast transformants with Y187(pGADT7-AGL24) respectively, did we find the five diploid yeasts can grow on QDO/X/A with blue color. It shows that the five mutant proteins of SOC 1 can interact with AGL24 protein, suggesting that the yeast fusion reporter genes HIS3, AUR1-C, ADE2 and MEL1 were activated.Yeast β-galactosidase activity analysis shows that the protein-protein interaction strength (Activity 21.58) between SOC1C137K mutant protein and AGL24 is significantly higher than AGL24 with SOC1 (Activity 4.44) indicating that the 137th amino acid residues of SOC1 can regulate polymerized strength of SOC 1 with AGL24. Moreover, there is no significant difference among the crosses SOC1V77K×AGL24, SOC1P81K×AGL24, SOC1K108V×AGL24, SOC1R109L ×AGL24 (Activity 3.14,2.73,2.50 and 3.19), but the activity of them is significantly lower than AGL24 with SOC1(Activity 4.44) showing that the 77th,81th,108th and 109th amino acid residues of SOC1 also can regulate polymerized strength of AGL24/SOC1.Meanwhile, we constructed SOC1 mutants fluorescence expression vector YNEOV77K, YNEOP81K,YNEOK108V, YNEOR109L and YNEOC137K respectively and transformed the recombinant plasmids into Agrobacterium GV3101 by Cold and hot stimulus. After determining positive clones, we mixed the broth with broth containing YCEG at equal amount and co-infiltrated 4 to 6 true leaves of N. benthamiana. After 48 h, we found that the guard cells and flagella of epidermal cells of the five combinations did fluoresce by laser confocal scanning microscopy at 488 nm excitation light, which indicates that they have interactions.