Cloning And Functional Characterization Of MsF-box Gene From Alfalfa

Low temperature is one of the major abiotic stresses affecting plant growth and development, and further limited agricultural production. F-box protein characterized by an F-box motif, a subunit of SCF complex, and works as substrate recognition region in the ubiquitin degradation pathway. F-box plays an essential role on abiotic stress response of plants. In the present study, a novel F-box gene was isolated from Medicago sativl L. and was designated as Ms F-box. Full length of Ms F-box gene was inserted into plant expression vector p CBM and transferred into Arabidopsis plants and tobacco plants mediated by Agrobacterium tumefaciens. Furthermore, physiological assays on the transgenic plants were analyzed to verified the resistance to cold stress.The summary of the results are present as follow:1. Cloning and silico analysis of Ms F-box geneMs F-box gene was cloned by RT-PCR. The full-length of Ms F-box is 1422 bp, and encoding 473 amino acid. The Ms F-box protein contains 1 F-box motif, and 6 leucine-rich repeats（LRR）domains. A BLASTp search indicated that Ms F-box showed high Identities with Mt F-box（Medicago truncatula, XP₀03626345）, the similarity is 96%.2. Construction of plant expression vector p CBM-Ms F-boxThe plant expression vector p CBM-Ms F-box was constructed. The ORF of Ms F-box was inserted into plant expression vector p CBM under the drive of 35 S promoter of Cauliflower mosaic virus（Ca MV）. The vector p CBM contain bar gene.3. Study of Ms F-box gene transformation in plant via Agrobacterium-mediated methods（1）Agrobacterium-mediated genetic transformation of tobaccoAgrobacterium EHA105 was transfomed by pCBM-MsF-box plasmid, and used to complete genetic transformation by infecting tobacco leaves. Tobacco leaves were trained in MS medium containing 1.1 mg/L PPT for screening cotyledon. PPT-resistant buds were removed for rooting to obtained transgenic plant.（2）Agrobacterium-mediated genetic transformation of ArabidopsisAgrobacterium EHA105 was transfomed by p CBM-Ms F-box plasmid, and used to complete genetic transformation by infecting Arabidopsis inflorescence. Putting harvested seeds into MS medium which containing 1.1 mg/L PPT for screening cotyledon and further obtained the transgenic lines.4. Molecular detection and cold resistance analysis of transgenic plantWe have developed 9 PCR-positive independent tobacco transgenic lines and 20 PCR-positive independent Arabidopsis transgenic lines. The transgenic lines（L8, L35 in tobacco and L8 in Arabidopsis） suffered from less severe stress under cold conditions.