Determination Of Interactions Between FLC And Homologous Truncated Forms In Brassica Juncea Coss. By Yeast Two-Hybrid System

Mustard (Brassica juncea Coss.) belongs to Cruciferae Brassica crops. Vernalization can induce plant flowering. FLOWERING LOCUS C (FLC), one of the key transcription factors which decide plant floral signal integrators, encodes a MADS-box transcription factor that represses flowering. Its expression is controlled by loci in the autonomous pathway and by vernalization. Both of the autonomous and vernalization can catch through the negative regulation of FLC expression, thus contributing to the flowering. The high levels expression of FLC inhibits downstream flowering integrated genes, and it has strong inhibition to flowering. FLC delays flowering by repressing production in the leaf of at least two systemic signals, reducing expression of these signals indirectly represses expression of genes involved in floral induction at the meristem. In addition, FLC expression in the meristem impairs response to the FT signal by directly repressing expression of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) MADS-box transcription factor and preventing up-regulation of the bZIP transcription factor FD. Repression of genes acting at multiple levels in this hierarchy is required for the extreme delay in flowering caused by FLC. In FRIGIDA (FRI) dependent pathway, FRI delays flowering by promoting FLC expression.MADS box transcription factors may interaction by each other to form protein complex, and participated plant regulation during its growth and development. In Arabidopsis thaliana, SVP, which also encoded a MADS box transcription factor, could interact with FLC to form protein complex. Mutation svp tremendously inhibited late-flowering phenotype by FLC. The protein interaction between mustard FLC and SVP were screened via pET prokaryotic expression system and yeast two-hybrid system they formed stable heterogeny protein complex and delay mustard flowering. Previous genetic and transgenic data had suggested that FLC interacted with itself or other MADS box proteins. But the study of FLC homologous interaction on mustard had not clear.For study on the molecular mechanism of flowering control in Brassica juncea Coss.(mustard), making mustard’QJ’for materal, the protein homologous interaction between FLC and truncated forms were screened via yeast two-hybrid system. The truncated genes of FLC1～5were respectively subcloned from yeast recombination plasmid pGBKT7FLC. The number of the amino acids encoded by FLC1～5was110,168,54,148and92. The proteins encoded by FLC1～5had MI-domain, MIK-domain, K-domain, IKC-domain and KC-domain, respectively. FLC1～5truncated forms were fused into prey plasmid pGBKT7, which were designated as pGBKT7FLC1～5and then transformed pGBKT7FLC and pGBKT7FLC1～5into Y2HGold stains. Also transformed pGADT7FLC into Y187yeast stains. All the transformed yeast stains were not autoactivation and toxicity. The yeast stains of pGBKT7FLC2～5and pGADT7FLC could mate into zygotes and grew on selective agar plates QDO/X/A with blue stains. Indicated that the report genes (AUR1-C, HIS3, ADE2, MEL1) of yeast stains were activated. The results showed that FLC2～5truncated forms and FLC protein could act with each other to form homodimers. It also indicated that K domain of FLC (FLC3) may plays an important role in mediating the FLC-FLC homodimers. Our result showed that:1Cloning of FLC and FLC1～5truncated forms genesWe designed specific primers to clone cDNA of FLC. The target gene FLC was amplified from Brassica juncea Coss.’QJ’stem apex cDNA by PCR. The gene magnitude of FLC was625bp. The sequencing consequence showed that the sequence of FLC was exactitude. Then the different truncated fragments FLC1～5were amplified from pEASY-Blunt Simple-FLC plasmid by PCR. The magnitude of FLC1～5genes fragments were341bp,513bp,198bp,466bp and301bp. By using1%agarose gel electrophoresis, the FLC and FLC1～5fragments length are identical with the theory value of primer extension. The sequencing consequence showed that the sequence of FLC1～5was exactitude.2The construction of yeast two-hybrid recombinant expression vectors By using molecular cloning technology, yeast two-hybrid recombinant bait plasmids pGBKT7-FLC, pGBKT7-FLC1, pGBKT7-FLC2, pGBKT7-FLC3, pGBKT7-FLC4and pGBKT7-FLC5and prey plasmids pGADT7-FLC were constructed. DNA sequencing consequence showed that pGBKT7-FLC and pGBKT7-FLC1-5recombinant bait plasmids contain FLC and FLC1～5fragments, pGADT7-FLC recombinant prey plasmid contain FLC fragment, and the insertion site and the reading frame were correct.3The toxicity and autoactivation detection of recombinant plasmidsThe transformants of Y2HGold[pGBKT7-FLC] and Y2HGold[pGBKT7-FLC1～5] were cultured on SD/-Trp plates at30℃. After three days, we can find white well clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-FLC and pGBKT7-FLC1～5were no toxic to yeast cell. The pGADT7-FLC was transformed into Y187, the ransformants of Y187[pGADT7-FLC] grew well on the SD/-Leu plates, while the yeast strain Y187[pGADT7] which was not transformed recombinant plasmid did not grow on SD/-Leu medium. It is suggested that recombinant plasmid Y187[pGADT7-FLC] was successfully transformed into Y187yeast cells and was not toxic to yeast cell Y187.In addition, Y2HGold[pGBKT7-FLC] and Y2HGold[pGBKT7-FLC1～5] could grow on SD/-Trp,SD/-Trp/x-a-gal plates and could not grow on SD/-Trp/x-a-gal/AbA plates, and the colonies did not turn blue on SD/-Trp/x-a-gal plates which indicates that pGBKT7-FLC and pGBKT7-FLC1～5don’t activate the reporter gene AUR1-C and MEL1in yeast cells. The white clones of Y187[pGADT7-FLC] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-FLC don’t activate the reporter gene MEL1.4The interaction detection between different truncated fragments of FLC and FLC, FLC and FLC1～5The recombinant plasmids were successfully transformed into the diploid yeast mating cell, the group of Y2HGold[pGBKT7-FLC]×Y187[pGADT7-FLC],Y2HGold[pGBKT7-FLC2]×Y187[pGADT7-FLC],Y2HGold[pGBKT7-FLC3]×Y187[pGADT7-FLC],Y2HGold[pGBKT7-FLC4]×Y187[pGADT7-FLC],Y2HGold[pGBKT7-FLC5]×Y187[pGADT7-FLC] could emerge different degrees blue clones on QDO/X/A plates, which might indicate that activate the reporter gene AUR1-C, HIS3, ADE2and MEL1.The results indicated: FLC1(M domain, I domain) couldn’t interact with FLC presumed that M domain and I domain hadn’t been the decisive effection in homologisation interaction; FLC2(M domain, I domain, K domain) could interact with FLC suggested that the homologisation protein complex were not decided by C domain; FLC3(only K domain) could interact with FLC suggested that K domain might mediate FLC form homologisation protein complex; FLC4(I domain, K domain, C domain) could interact with FLC suggested that the homologisation protein complex were not decided by M domain; FLC5(K domain, C domain) could interact with FLC suggested that the homologisation protein complex were not decided by I domain.To sum up, no matter M domain, I domain or C domain, it couldn’t mediate FLC homologisation protein interaction, K domain might play a decisive role in mediating FLC homologisation protein interaction.