Cloning And Functional Characterization Of MsMADS1 Gene From Alfalfa ?Medicago Sativa L.?

Low temperature is one of the major abiotic stresses that limit plant growth and crop yield.MADS-box protein which plays an important role in regulating the growth and reproduction of plants is one of the largest widely studied transcription factor family in plants.In this study,Ms MADS1 was cloned from alfalfa?Medicago sativa L.?and its plant expression vector was constructed.Then,the Ms MADS1 gene was transferred into the tobacco plant to study the function of Ms MADS1 gene.The main results are as follows:1.Cloning and sequence analysis of MsMADS1 geneThe cloning primers were designed according to the sequence information of transcriptome of alfalfa under the low temperature stress,and the MsMADS1 gene was cloned by RT-PCR.The length of the gene was 741 bp,encoding 246 amino acids.The protein contains a MADS-box domain and a K-box domain.The NCBI protein BLAST showed that the amino acid similarity of MsMADS1 and Mt MADS-box was 90%,and there were three different sites in the domain.2.Relative expression of Ms MADS1 geneThe relative expression of Ms MADS1 gene was detected by qRT-PCR under different tissue and abiotic stresses.The results showed that Ms MADS1 gene was expressed in roots,stems,leaves,flower and fruit,with the highest expressed level in roots.Under the different abiotic stresses,Ms MADS1 gene in leaf and root was not only strongly induced by low temperature stress but also response by salt,saline-alkaline,drought and ABA.3.Construction of plant expression vector pCAMBIA1305.1-Ms MADS1The plant expression vector PCAMBIA1305.1-Ms MADS1 was constructed,which contained Ca MV35S promoter and hygromycin resistance.4.Transformation of Ms MADS1 gene in tobacco plantMsMADS1 gene was transferred into the tobacco plant by Agrobacterium mediated method,and 35 resistant plants were obtained by screened.A total of 21 transgenic plants were identified by PCR.The result showed that the three lines of 4,18,29 had a higher expressed level than other transgenic lines and control plants by qRT-PCR,and these chosen lines would be studied further.5.Analysis of cold resistance in Ms MADS1 transgenic tobaccoThe transgenic tobacco was treated under low temperature stress in order to analyze the cold resistance of transgenic tobacco.Under low temperature treatment,wild type tobacco was severely injured and completely wilted,but the injury of MsMADS1 transgenic tobacco was lighter.Under low temperature stress,the electrolyte leakage,MDA content,O₂^-content and H₂O₂ content of transgenic tobacco were lower than that of wild type.Soluble protein content,proline content and antioxidant enzyme activities were higher than that of wild type.This results suggest that MsMADS1 gene was involved in plant tolerance to cold stress.