Construction Of Transients System And Function Analysis Of Flowering Locus T Gene In Mulberry(Morus Alba.L)

FT gene is a promoting factor in flowering signaling pathway,and collection point of flower developmental pathways,integration flower developmental signals from the photopefiodic pathway,vernalization pathway and autonomous pathway.FT is the key signaling factor for determination of flowering time in higher plant.FT gene not only adjust the phase shift of stage child to adult, also control plant growth rhythm,conversion plant growth and dormancy,conversion the cycle of vegetative growth and reproductive growth in plant.It takes 3-5 years from sowing to flower in mulberry.Long infancy is the biggest obstacle to genetic breeding research and also the main factors which influence the production of early mulberry fruits.MaFT is the floral factor in mulberry.So far, no studies were reported on genes expression and function of MaFT family.In this paper,we studied on MaFT gene,around the function of MaFT gene in transient expression and grafted mulberry and function in transgenic arabidopsis and alfalfa.The mainly results as follows:1.We obtained recombinant FT protein via prokaryotic expression veror pET28a-MaFT. MaFT protein is the form of inclusion body.We purified protein by using His label and renaturated protein by urea gradient method through the dialysis membrane.The protein as antigen was fused with Freund’s adjuvant and incomplete Freund’s adjuvant to immunize rabbit for preparing polyclonal antibody.Four times after immunization in the New Zealand rabbit, we obtained efficient antibody with titer of 1:512,000.2.A plant binary expression vector named pBE2113-FT-GFP harboring both mulberry FT gene and GFP reporter gene was constructed and transformed into Arabidopsis thaliana by flower dipping method.Transgenic Arabidopsis thaliana plantlets were obtained through screening by kanamycin resistance and RT-PCR,Western blot analysis of total proteins. Results demonstrated that MaFT gene had been integrated into genome of Arabidopsis thaliana and successfully expressed.It was found that the transgenic Arabidopsis thaliana flowered earlier 10 days than wild type, at 25 d after the transgenic Arabidopsis thaliana seeds were sown, the plants began to flower.At this time, the plants had only sprouted out 5 young leaves.These dates verify that MaFT gene can lead to early blossoming in Arabidopsis thaliana.3.To optimize Agrobacterium-mediated transient transformation assay in mulberry.Various infiltration methods, Agrobacterium tumefaciens strains,and bacterial concentrations were tested in mulberry seedlings.Compared with LBA4404,GV3101 harboring pBE2113 plasmids presented stronger GUS signals at 3 days post infiltration using syringe.Results showed that GV3101 agrobacterium strains are more suitable for transient;an efficient transient transformation system could be achieved in mulberry seedlings by syringe using Agrobacterium tumefaciens GV3101 at the OD600 of 0.5;The meat thick fresh leaf than old leaf is more conducive to improving the efficiency of transient;tissue culture seedings and seedings has a little influence on transient expression.Recombinant plasmids pBE2113:GFP and pBE2113:MaFT:GFP were successfully constructed.Transient expression of MaFT:GFP protein was found in leaves,petiole(cross section) and shoot apical meristem(SAM) of mulberry according to the GFP signal.Moreover,MaFT:GFP mRNA was also detected in leaves via qRT-PCR,analysis showed that FT expression quantity highest by day 4 after transient expression,then declining gradually;western blot test showed that the FT protein can conduct through the phloem from the leave to the SAM.4. The expression of FT and protein conduction rule was detected in grafted mulberry. Analysis change levels of FT mRNA between grafted and non-grafted seeding mulberry leaves and apical buds via qRT-PCR, grafted and non-grafted seeding mulberry were sowed in the same year;results showed that low relative expression of FT in non-grafted seeding mulberry and FT expression rised a little after grafted three months in mature leaves,Within one year after grafted, FT expression is more two times than three months grafted mulberry leaves,is three times than non-grafted seeding mulberry leaves.Within two years,FT expression increased was not obviously in non-grafted seeding mulberry leaves.We detected FT protein in leaves and apical buds via Western blot after grafted three months and one year respectively,FT protein was only detected in mulberry leaves.These results showed that the FT gene expressed mainly in leaves and its protein can be transmitted from donor mulberry to receptor mulberry.5. Agrobacterium-mediated genetic transformation system of alfalfa was optimized. Co-cultured for 3 days, agrobacterium cell density was 0.6 at OD600 for a 5 min inoculation and the mediums containing 50 mg/L kanamycin and 500 mg/L carbenicillin for induction of kanamycin-resistance callus were the best conditions for transformation.MaFT gene was transformed into alfalfa and resistant alfalfa shoots were identified via PCR, up of FT and down of GFP primers were used for PCR.Results showed that MaFT has been transformed into alfalfa’s genome.This study has an important theoretical significance and application value to know molecular regulation mechanism of flowering, promote early fertility fruits, regulate FT gene expression, shorten juvenile phase and improve the efficiency of breeding in mulberry.We know flowering is a complex process, regulated by various pathways such as plant itself, environmental factors and other ways and limitation on the transgenic technology in mulberry. Our research is still in the primary stage, the MaFT gene function of regulation mechanism in flowering and dormancy has yet to be further studied.