Study On Interactions Between FLC And SVP In Brassica Juncea Coss

Flowering is one of the most important phase changes during the vegetative to reproductive growth in the life cycle of higher plants. In the plant, the early-or late-flowering may be brought a lot of negative factor in the breeding or seeding production F1. In Brassica juncea Coss, the time of the flowering is determined by the intricate network of pathway integrating endogenous and environmental (such as temperature and light). To similar with Arabidopsis, there are four genetic pathways to regulate the mustard flowering:vernalization pathway, autonomous pathway, photopenod pathway and gibberellin pathway. And the four genetic pathways are converge on floral pathway integrators, which are some key genes to integrate inputs from the different flowering signals. FLOWERING LOCUS T (FT), SUPPRESSOR OF CO OVEREXPRESSION1(SOC1), FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE(SVP)have been identified as such integrators in Arabidopsis, FT and SOC1acts as promoter for flowering. FLC and SVP are negative of flowering in higher plant. The mustard act as an important Brassicaceae vegetable has a wide range of plant area in south China, and it belongs to the seed vernalization plant. Therefore, it is necessary to study the flowering time of mustard for production and practice.In this research, FLC and SVP gene were isolated by using degenerate primers from the mustard cDNA, they acts as negative regulator of flowering in Arabidopsis. Subsequence, using yeast two-hybrid system to study their potential interaction domains between FLC-SVP protein complex. The main results were showed as follows:According to the conserved motif of cloned genes of FLC and SVP in Arabidopsis and other Brassicaceae plant, the degenerate primers were used to amplify the two genes from shoot apex cDNA in mustard. The FLC gene has an open reading frame of759bp and encoding a MADS-box protein of197amino acids, phylogenetic study show that it is a close relationship with the Brassica oleracea var. capitata FLC(AAQ76273). The full-length cDNA of SVP gene has an757bp and encoding a protein of241amino acids, phylogenetic study indicated that it is a close relationship with the Brassica rapa subsp. Campestris SVP(AAQ55451). There two genes were belongs to repressor of flowering in Brassicaceae.Using the homologous recombination technology, constructed a group of pGADT7FLC and pGBKT7SVP recombinant vectors that were subsequently transformed accordingly into Y187and Y2HGold yeast. The zygote diploids grew on selective agar plate QDO/X/A(SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA) with blue stains. Furthermore, the vectors of bait plasmid and prey plasmid were exchanged with each other. The yeast zygote diploids(Y187(pGADT7SVP) xY2HGold (pGBKT7FLC)) exhibit on selective agar plates QDO/X/A. The results showed that FLC protein interacted with SVP protein in vivo.We subcloned the truncated genes of FLC1～5and SVP1～5from full length FLC and SVP gene, and all of the truncated genes were inserted into the yeast two hybrid expression vectors. The proteins encoded by SVP1～5or FLC1～5had MI-domain, MIK-domain, K-domain, IKC-domain and KC-domain, respectively. The yeast zygote diploids of Y187(pGADT7SVP2-5) x Y2HGold(pGBKT7FLC) and Y187(pGADT7SVP) x Y2HGold(pGBKT7FLC2-5) were able to grew on selective agar plates QDO/X/A with blue stains. The results show that K domain was the key amino acid region to independently mediate the interactions between FLC and SVP. To further confirm the acting domains between FLC and SVP, the interactions between SVP3and FLC3were tested. The zygotes Y187(pGADT7SVP3) x Y2HGold(pGBKT7FLC3) and Y187(pGADT7FLC3) x Y2HGold (pGBKT7SVP3) exhibit blue stains on selective agar plates QDO/X/A. The results finally showed that the K domain act a key structure domains to mediated the FLC-SVP protein dimerization.