Identification Of Protein Interactions Between BjSVP From Brassica Juncea And BoFLC From Brassica Oleracea

Cabbage (Brassica oleracea L.) and mustard (Brassica juncea Coss.). which belong to Cruciferae Brassica crops, are widely grown in China. Vernalization treatment can induce plant flowering. Among them, the mustard belongs to the seed vernalization plant that can induce feelings of low temperature induction in a seed germinating state; while the cabbage is vernalization body plant, which need to grow to a certain size, the accumulation of certain nutrients before they can feel the low temperatures induction. The difference was the feeling low-temperature in different parts:feelings of low-temperature parts in the embryo during seed germination, and in the shoot tips during the growing phase. Flowering is one of the transition phase from the vegetative growth to reproductive growth to marke the plant throughout the life cycle. For the plants, the early-or late-flowering may be brought a lot of negative factors in the breeding or seeding production F1. Therefore, it is of great significance to study the flowering time of mustard and cabbage for production area to expand cultivation and practice to breed quality varieties.In similar with Arabidopsis, there are many genetic pathways to regulate the mustard and cabbage flowering:vernalization pathway, autonomous pathway, gibberellin pathway, photoperiod pathway and temperature-sensitive pathway. These pathways include various flowering regulatory signals, they are converge on floral pathway integrators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1). FLOWERING LOCUS C(FLC and SHORT VEGETATIVE PHASE (SVP) are two typical MADS protein, flowering regulatory network formed by the protein interaction between SVP and FLC, that led to suppress flowering signal integrators expression, so late-flowering. Currently, identification of protein could interact between FLC and SVP in the seed vernalization plant of Arabidopsis, mustard and the green vernalization plant of cabbage. However, previous studies have focused on the presence of homologous plant from FLC and SVP interaction, and the interaction of FLC and SVP in different crops has not been reported.In order to clarify the cabbage BoFLC heterologous protein interaction mechanism of the mustard BjSVP. the mustard ’QJ’and cabbage’ZQ’ as experimental material, BoFLC, BjSVP and BjSVP truncated body(BjSVP1～BjSVP11) genes were cloned, yeast recombination plasmids including the three genes respectively were built via the yeast two-hybrid technology, we studied their potential interaction domains between FLC-SVP protein complex, and analyzed of major protein-protein interaction domain.1、BoFLC. BjSVP and BjSVP truncated froms genes were cloned and analyzed.According to the structural characteristics and identifying characteristics of FLC and SVP gene, specific primers were designed. The target genes were amplified from Brassica oleracea L.’ZQ’ and Brassica juncea Coss.’QJ’stem apex cDNA by PCR. The full-length cDNA of FLC encoding amino acid residues197was625bp. the gene magnitude of SVP encoding a protein of241amino acids was757bp. The PCR amplification, restriction enzyme digestion and sequencing consequence have showed that full-length gene sequence was exactitude. Then the different truncated fragments BjSVP1～11were subcloned from pEAST-Blunt Simple-BjSVP plasmid by specific primers, encoding95,173,78,179,146,111,86,80,64.52and33amino acids. PCR amplification, restriction enzyme digestion and sequencing consequence have showed that BjSVP1～11gene sequence was exactitude.2、The interaction between BjSVP and the full length BoFLC was identificated.Making use of the yeast two-hybrid system, the bait plasmid pGBKT7-BoFLC was built, and then transformed into Y187and Y2HGold yeast. Results showed that Y2HGold activity was stronger than Y187, and the transformants of Y2HGold (pGBKT7-BoFLC) and Y187(pGADT7-BjSVP) on less selective media grew normally, which showed that the recombinant bait plasmids were no toxic and autoactivation detection phenomenon to yest cell. Fusion diploid yeast Y187(pGADT7-BjSVP)x Y2HGold (plasmids pGBKT7-BoFLC) can be selected from medium SD/-Leu/-Trp/AbA(DDO/A), SD/-Ade/-His/-Leu/-Trp(QDO), SD/-Ade/-His/-Leu/-Trp/Xa-Gal/AbA(QDO/X/A). And the clones grown in QDO/X/A medium showed blue. The results showed that the the full length SVP cloned from the mustard and FLC from the cabbage interaction to form a dimer complex, which activated the reporter gene AUR1-C,HIS3, ADE2, MEL.3、The protein interaction domains of BoFLC and BjSVP was screened and identificated.The proteins encoded by BjSVP1-11respectively had MI, MIK, K, IKC. KC, IK, IK1L1K2L2, IK1L1K2, IK1L1, IK1or I domains. BjSVP1-11truncated forms were fused into prey plasmid pGADT7, which were designated as pGADT7-BjSVP1-11and then transformed into Y187yeast stains. The yeast transformation Y187(pGADT7-BjSVP1～11) in deficient selective medium was identified, and the phenomenon of self-activation and toxicity were not discovered. The diploid recombinant yeast containing fusion after only the group of Y187(pGADT7-BjSVP2～6) X Y2HGold (pGBKT7-BoFLC) was blue on QDO/X/A plates. The result showed that the truncated BjSVP2～BjSVP6can interact with BoFLC, BjSVP7～BjSVP11can not interact with BoFLC. which indicated that K domain of BjSVP (BjSVP3) was the key amino acid region of mediating the interactions between BjSVP and BoFLC independently. However, BjSVP truncated forms with deletions of subdomains (K1, K2or K3) or linkage regions (L1or L2) in K domain could not act with BoFLC. Analysis of the strength of the effect showed that:I domain of BjSVP could strengthen the interaction of proteins. But M and C domain of BjSVP could probably weaken the interaction of proteins.