Cloning And Function Analysis Of A Zeaxanthin Epoxidase Gene（MsZEP）from Alfalfa（Medicago Sativa.L）

Alfalfa(Medicago sativa.L), an important leguminous herbages, can ?x nitrogen by interaction symbiotically with rhizobia in root nodules. Because of its wide range of cultivation, strong adaptability, high forage quality and high yield, alfafla was praised as the "king of forage". Both the growth of legume plants and the nodules functions are limited by drought, salt, high temperature. With the development of biotechnology, alfalfa breeding technology has turned from traditional breeding to molecular ways. Recent year, studies on gene cloning, functional verification and genetic transformation of alfalfa developed very fast.In this study, MsZEP cDNA was cloned by rapid-amplification of cDNA ends(RACE)technique, and the sequence was analyzed by using bioinformatics software. Moreover, the promoter of MsZEP was isolated and the cis-elemennts were analyzed. The expression patterrn of MsZEP under different abiotic stress treatments were analyzed by qRT-PCR. In addition, two fusion protein expression vectors, pCAMBIA1300-MsZEP and an RNAi vector MsZEP-RNAi, were constructed. Then the vector pCAMBIA1300-Ms ZEP was transformed into tobacco and alfalfa by Agrobacterium. Futhermore, MsZEP-overexpression tobacco was used to analyze the fuction of MsZEP.The main results were as following:1. The complete cDNA sequence of MsZEP was amplified from alfalfa by RACE-PCR.Sequencing analysis revealed that the full-length open reading frame of Ms ZEP contains 1992 bp nucleotides and encodes a protein of 663 amino acid residues, and the Genbank accession number was KM044311.2. The promoter sequence of MsZEP gene was obtained using Genomic DNA walking method. Sequencing analysis revealed that the full-length of the promoter contains 995 bp nucleotides, and the promoter contains several light sensing elements, stress-responsive elements, hormone-responsive elements, TATA boxes and CAAT boxes.3. qRT-PCR was performed to reveal the transcript patterns of MsZEP in different tissues and under different stresses: the results indicated that MsZEP transcription was abundant in green tissues(leaves and stems); MsZEP expression decreased in shoots under drought, cold,heat and ABA treatment, while the expression levels in roots showed different trends. Besides,the results showed that nodules could up-regulate the MsZEP expression under normal conditions and in the earlier stage of abiotic stress.4. We constructed three fusion protein expression vectors pCAMBIA1300-MsZEP,MsZEP-GFP and MsZEP-RNAi. We transformed MsZEP-GFP into tobacco leaves.Subcellular localization result showed that MsZEP was deposited into chloroplast.5. The vector pCAMBIA1300-MsZEP was transformed into tobacco or alfalfa by Agrobacterium mediated transformation. Hygromycin-resistant tobacco and alfalfa plants were obtained. PCR and RT-PCR analysis showed that MsZEP gene was transformed into regenerated tobacco or alfalfa.6. The MsZEP gene enhanced salt and drought tolerance of transgenic tobacco by changing various physiological pathways, ABA levels, stomatal aperture and stress-responsive genes.7. MsZEP gene negatively regulated seed germination by increasing seeds ABA level.The increased ABA content was associated with an up-regulation of transcript levels of ABA biosynthetic genes.8. After weak light treatment, MsZEP-overexpression tobacco exhibited higher fresh weight, more leaves, bigger leaf size and stronger photosynthetic ability than wild type tobacco.