Cloning And Functional Analysis Of Gibberellin Receptor Gene MsGID1b From Medicago Sativa L.

Alfalfa（Medicago sativa L.）is a kind of perennial legume forage with high quality.Increasing its biomass yield has been one of the main breeding objectives.Plant biomass has been effectively improved by overexpression several main components of Gibberellin biosynthetic pathway and signaling pathway.Gibberellin receptor（GID）plays an important role in gibberellin signal transduction pathway,which directly affects plant growth and development.In this study,a gibberellin receptor gene,MsGID1b,was first isolated from Medicago sativa L.by homologous cloning.Sequence analysis showed that MsGID1b contains 350 amino acid residues with 39.839 kD molecular weight,and has high nucleotide（98%）and amino acid（99%）similarity with its orthologous gene in Medicago truncatula.Physical and chemical properties analysis defined that MsGID1b was a kind of hydrophilic protein without signal peptide and transmembrane structures.Protein subcellular localization server indicated that MsGID1b was predominantly located in the cytoplasm and microbody.MsGID1b contains the binding site of GA,DELLA and several conserved domain of hormone-sensitive lipase family（HSL）like HGG and GXSXG.Spatio and temporal expression profile investigated by real-time PCR indicated that MsGID1b expressed comprehensively with its highest expression level in root.MsGID1b was sensitive to GA₃,ABA,Na Cl,PEG and dark treatments,and it maintained a high transcript level under GA₃ treatment,suggesting that MsGID1b might participate in the response of abiotic stress of Medicago sativa L..SDS-PAGE showed that the recombinant plasmid pEASY-MsGID1b was expressed successfully in E.coli DE3.Moreover,the expressed protein size was consistent with the predicted.Yeast Two-Hybrid experiment showed that MsGID1b can interaction with At RGA and At GAI,two main DELLA protein of Arabidopsis thaliana,in the presence of GA₃.An overpression recombinant plasmid,pBI121-MsGID1b,was constructed successfully and transferred into Arabidopsis thaliana by agrobacterium-mediated transformation.Transgenetic lines was identified by antibiotic resistance screening and molecular testing.Plant height,dry and fresh weight,pod number has increased in transgenic Arabidopsis.Particularly,the number of pods is twice that of the control group.Plant weight,including dry and fresh weight,has increased significantly.It was suggested that MsGID1b has a certain effect on the growth and development of plant,especially for flower and fruit development.