Subdomains Of K Domain Mediates Heterodimerization And Homodimerization Of Brassica Juncea Flowering Time Regulators,FLC And SVP

Blossom is the conversion of plants from vegetative growth stage to reproductive growth, which is an important developmental stage during the life cycle of plants. It decided the feasibility of sexual reproduction of plants. The timing of the blossom determines the time major plants products like fruits and seeds come into the market. Therefore, to do the plants blossom researches has fundamental economic value for both production and breeding of plants. As a very important cruciferous vegetable crop, Brassica juncea has a certain amount of acreage around the country. The time of blossom is affected by internal conditions such as the state of plants growth, the developmental stage and genetic factors, in addition to the effect of external environment like the light (such as the intensity and the length of sunlight) and the temperature. There are four pathways to regulate the blossom of mustard, which are autonomous, vernalization, photoperiod response and GA pathway. Even though those four pathways have their own downstream gene regulatory networks, they all finally converge to the blossom integron to finish the blossom regulation. Until now, a large amount of genes that are related to the bolting and blossom of plants have been found, such as FLOWERINGLOCUS T (FT), SUPPRESSOR OF CO OVEREXPRESSION1{SOC1),FLOWERINGLOCUS C (FLC),SHORT VEGETATIVE PHASE(SVP). Among them, FT and SOC1function in promoting the blossom while FLC and SVP suppress the blossom. SHORT VEGETATIVE PHASE (SVP) gene was first identified in Arabidopsis, which is the first to be confirmed through En-1transposon, located on the second chromosome, on early flowering mutants. The SVP protein is a typical MIKC-type protein, containing a highly conservative MADS domain and K domain, as well as relatively weaker conservative I domain and C domain. The previous research of our laboratory had fuond:Brassica juncea, its SVP encoded protein is still the protein of MIKC-type MADS domain. FLC is a crucial blossom inhibition MADS-box gene, whose encoded protein also belongs to MADS-box transcription factor. The previous research of our laboratory had fuond that a dimeric complex is formed through the interaction between FLC and SVP protein in mustard Then it mediates the signal from environment and inhibits the expression of the downstream FT and SOC1blossom integron, thus inhibiting the plants blossom. We also found that, K domain is the interaction domain of FLC and SVP, but K-domain can be divided into K1, K2, K3exactly three sub-domains which sub-domain protein interactions play a decisive role remains unclear. Therefore, this experiment screen FLC and SVP sub-domain interactions of Brassica juncea.1、The Brassica juncea FLC and SVP subdomains cloning and the construction of yeast carriersIn this study, laboratory preserved Brassica juncea yeast recombinant plasmid pGADT7-FLC, pGADT7-SVP, pGBKT7-FLC, pGBKT7-SVP sequences were served as references for primer design. These sub-cloned were:BjSVP (MIKC) encoded241amino acids, BjSVP△1(I) encoded57amino acids, BjSVP△2(K) encoded78amino acids, BjSVP△3(IK) encoded111amino acids, BjSVP△4(IK1L1K2L2) encoded84amino acids, BjSVP△5(IK1L1K2) encoded80amino acids, BjSVP△6(IK1L1) encoded64amino acids, BjSVP△7(IK1) encoded52amino acids; BjFLC (MIKC) encoded197amino acids, BjFLC△1(I) encoded57amino acids, BjFLC△2(K) encoded54amino acids, BjFLC△3(IK) encoded115amino acids, BjFLC△4(IK1K2) encoded108amino acids, BjFLC△5(IK1) encoded78amino acids. We also constructed yeast two-hybrid recombinant expression vector of each fragment and analyze specific zones where K domain subdomain-mediated heterologous and homologous proteins interacted.By applying the homologous recombinant technology, each various FLC and SVP truncated yeast expression vector, such as pGBKT7-BjFLC, pGBKT7-BjFLC△1-5,pGBKT7-BjSVP,pGADT7-BjSVP, pGADT7-BjSVP△1-7, and PGADT7-BjFLC, was constructed. The recombinant yeast plasmid was transformed into competent yeast through the lithium acetate transformation method, obtaining yeast transformants Y2Hgold[pGBKT7-BjFLC], Y2Hgold[pGBKT7-BjSVP], Y2Hgold[pGBKT7-BjFLC△ 1～5] and Y187[pGADT7-BjSVP], Y187[pGADT7-BjFLC], Y187[pGADT7-BjSVP△1～7]. During the toxicity testing, the transformants transferred into the pGBKT7recombinant vector showed that recombinant plasmids have no toxicity for Y2Hgold bacterial strain after the SD/-Trp screening. And the SD/-Leu screened transformants transferred into the pGADT7recombinant vector showed no toxicity for Y187bacterial strain. In the self-activation assay, transformants transferred into pGBKT7recombinant vectors went through the narrowing down screening of SD/-Trp, SD/-Trp/X-a-Gal, SD/-Leu/-Trp (DDO), SD/-Trp/Xa-Gal/AbA substrate, and it turned out that the self-activation of each transformant did not exist in Y2Hgold strains; Those transformants transferred into pGADT7recombinant vector after going through the narrowing down screening of SD/-Leu, SD/-Leu/X-a-Gal, SD/-Leu/-Trp drill (DDO) and SD/-Leu/X-a-Gal/AbA substrate screening showed that none of the transformants have self-activating phenomenon in Y187strains.2、The interaction between K domain subdomain-mediated FLC and S VP heterologous proteinsY187[pGADT7-BjSVP△1～7] combined with the Y2Hgold[pGBKT7-BjSVP] transformation strains respectively. Among the combined yeasts, Y187[pGADT7-BjSVP△2～7]×Y2HGold[pGBKT7-BjFLC] and Y187[pGADT7-BjSVP] xY2HGold[pGBKT7-BjFLC] plates could have the blue colonies grow on them. Therefore, it is supposed that during the interaction between FLC and SVP heterologous protein, the K1subdomain of the BjSVP protein’s K domain is the combination zone of the interaction. The β-galactosidase activity detection and analysis of variance presented that:During the interaction of heterologous proteins, the K2subdomain in BjSVP K protein could enhance the protein interaction while the K3subdomain and L1intersection had no influence on protein interacting intensity. However, the L2intersection could undermine the interaction.The combined strain yeast transformant Y2HGold[pGBKT7-BjFLC△1～5] integrate with transformant Y187[pGADT7-BjSVP△7] respectively, and then blue colonies of the combined strain Y2HGold[pGBKT7-BjFLC] xY187[pGADT7-BjSVP△7] and Y2HGold[pGBKT7-BjFLC△2～5] xY187[pGADT7-BjSVP△7] appear on the SD/-Trp/-Leu/-Ade/-His/X-a-Gal/AbA (QDO/X-a-Gal/AbA) plate. Therefore, it is conjectured the K1subdomain of the K domain in BjFLC protein is the combination zone of the interaction. The β-galactosidase activity detection and analysis of variance presented that:The K2subdomain of K domain in FLC protein could intensify the interaction while K3subdomain did the opposite.3、The interaction between K domain subdomain-mediated SVP and FLC homologous proteinsIn the SVP and SVP homologous protein interaction domain screening tests, transformant Y187[pGADT7-BjSVP△1-7] and Y2Hgold[pGBKT7-BjSVP] integrate respectively. Then the fused strain [pGADT7-BjSVP△2]×Y2HGold[pGBKT7-BjSVP]、 Y187[pGADT7-BjSVP△3]×Y2HGold[pGBKT7-BjSVP] have the blue colonies grown on the SD/-Trp/-Leu/-Ade/-His/X-a-Gal/AbA (QDO/X-a-Gal/AbA) plates. Based on that, the conjecture is that a complete K domain (K1L1K2L2K3) is necessary for BjSVP’s self-interaction when SVP homologous proteins interact.In the FLC and FLC homologous protein interaction domain screening tests, transformant Y2Hgold[pGBKT7-BjFLC△1-5] and Y187[pGADT7-FLC] integrated respectively. Then the fused strain Y2Hgold[pGBKT7-FLCA2-5]xY187[pGADT7-FLC] all have the blue colonies grown on SD/-Trp/-Leu/-Ade/-His/X-a-Gal/AbA (QDO/X-a-Gal/AbA) plates. Therefore, it is supposed that during the FLC homologous protein interaction, the K domain, IK domain, IK1K2, IK1of BjFLC could all interact with the BjFLC protein itself. Meanwhile, K1subdomain is the key area of BjFLC homologous interaction.The results of β-galactosidase activity detection and analysis of variance test showed that:During the homologous interaction of the BjFLC protein, K2and K3subdomain would attenuate the protein interaction intensity.