Molecular Cloning And Functional Analysis Of Msduf From Medicago Sativa

Salinity and drought are two major abiotic factors that limit crop production and forage yield, especially in the arid and semi-arid areas of northwest China. Alfalfa (Medicago sativa L.) is one of the most important legume forages and plays a very important role in the agriculture and animal husbandry, and the ecological construction in the arid and semi-arid areas of northwest China. Alfalfa is a medium salt-tolerant plant, but not for long-term cultivation in saline soil. Breeding enhanced salt and drought resistant alfalfa cultivars is one of necessary ways to increasing forage yield of alfalfa pastures, in the arid and semi-arid areas of northwest China. With the development of biotechnology, genetic engineering technique is playing a vital role in studies on enhancing drought, salt and cold tolerance of plants.In this study, we cloned MsDUF by rapid-amplification of cDNA ends(RACE), and constructed overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. In order to obtain more stress-tolerant tobacco and alfalfa materials, relative physiological indicators were determined to verify the effectiveness of MsDUF gene in improving plant tolerance to stress.The main results were as following:1.The complete coding cDNA of MsDUF was amplified from Medicago sativa pods mRNA by rapid-amplification of cDNA ends(RACE). Sequencing analysis reveals full length of open reading frame of MsDUF was633bp and encoded a protein of210amino acids residues, and the Genbank accession number is JX183734.2.Real-time PCR was performed to reveal that transcript level of MsDUF in different tissues and under different stresses. The results indicated that MsDUF transcription was abundant in roots, and the expression of MsDUF was induced by NaCl and PEG treatments.3.We constructed the fusion protein expression vector PBI221-GFP, and transformed it into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed MsDUF-GFP fusion protein was detected by a confocal laser scanning microscope. The result revealed that MsDUF was localized in cytoplasm. 4.The coding region of MsDUF was inserted into the expression vector pCAMBIA1301to constructed the recombined overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. Ten kanamycin-resistant tobaccos and alfalfa were obtained. PCR and RT-PCR analysis showed that8and7plants were detected target fragment in tobaccos and alfalfa.5.To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. The relative water content (RWC), malondialdehyde (MDA) content, soluble sugar and proline content were measured. Results showed that the MDA content was significantly lower in the transgenic tobaccos that in wild ones. However, the RWC, MDA and proline content accumulated much more in transgenic tobaccos than in wild plants. It demonstrates a role of the MsDUF protein in stress protection and suggests the potential of the MsDUF gene for improving the salt stress tolerance of tobacco and alfalfa.