Cloning And Function Analysis Of A Cytokinin Oxidase Gene(MsCKX) From Alfalfa(Medicago Sativa.L)

Alfalfa(Medicago sativa.L)plays an important role in the development of animal husbandry as a high quality legume forage.Alfalfa has a large area of cultivation in China,of which the Northwest region is the main planting area.However,the yield and quality of alfalfa have been greatly influenced by the low rainfall and the soil salinization of northwest region.Therefore,it is imperative to breed alfalfa varieties with stronger resistance to adversity.Studies have shown that cytokinin oxidase gene acts as an adversity response factor to participate in plant stress response.Therefore,study the function of cytokinin oxidase gene and control the endogenous cytokinin level by artificial means,which is an effective biotechnological strategy for improving the resistance of alfalfa.In this study: the cytokinin oxidase gene of alfalfa(MsCKX),was cloned by homologous cloning,the sequence and molecular functions were analyzed by bioinformatics software;the expression patterrn of MsCKX under different abiotic stress treatments were analyzed by real-time fluorescence quantitative PCR(q RT-PCR);the overexpression vector pCAMBIA1302-MsCKX was constructed and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens.Then,transgenic Arabidopsis was used to analyze the elementary fuction of MsCKX.The main results are as follows:1.The full-length of the MsCKX gene was 1833 bp,of which open reading frame contains 1530 bp nucleotides and encodes a protein of 509 amino acid residues.Signal peptide prediction indicates that MsCKX protein does not contain signal peptide structure,and may function in cytoplasm.The conservative domain analysis showed that MsCKX contained the core functional domains of the CKX family: cytokinin dehydrogenaseI,FAD and cytokinin binding,FAD/FMN-containing dehydrogenase and FAD-linked oxidoreductase.2.MsCKX transcription was appeared in leaves and roots,but the highest expression was in the leaves and it was 16 times as root expression;the expression of MsCKX gene varies with different tissues and environmental stimuli,and the expression pattern and amount was large after drought,salt and ABA treatments.However,no matter what changes,MsCKX gene responds to all kinds of stresses,indicating that MsCKX gene may participate in the regulation process of ABA,salt and drought stress.3.Hygromycin-resistant Arabidopsis thaliana were obtained and the transgenic Arabidopsis was used to analyze the elementary fuction of MsCKX from two aspects as seed germination and abiotic resistance(drought resistance and salt tolerance).It showed that the MsCKX gene had a negative regulation effect on the seed germination of Arabidopsis thaliana,delayed its germination and increased its salt tolerance and drought resistance by extending the root length.In summary,the preliminary function of MsCKX gene in alfalfa was verified through bioinformatics,expression patterns and transgene analysis.It laid a foundation for the study of alfalfa resistance.