Cloning,expresion And Transformation Of A Peroxidase Gene(MsPrx) From Alfalfa(Medicago Sativa.l)

Alfalfa is an important legume forage,with high protein content,good palatability,high yield.Alfalfa has a large area of cultivation in China,of which the Northwest region is the main planting area.However,due to the low rainfall in the northwest region and the soil salinization,alfalfa is often affected by these adverse environments which greatly affect the yield and quality of alfalfa.Therefore,it is of great significance to obtain more resistant varieties of alfalfa to promote the development of animal husbandry in China.However,because of the low efficiency of traditional breeding methods,the traditional means did not have a great breakthrough.Transgenic technology has the advantages of high efficiency and strong purpose.Therefore,it is very effective and feasible to improve the resistance of alfalfa by molecular technique.In this study,the peroxidase gene of alfalfa,MsPrx,was cloned by homologous cloning,and its molecular function was speculated by gene and protein sequence analysis.The different tissues expression of MsPrx gene were analyzed by real-time fluorescence quantitative PCR,as well as treatment with NaCl and ABA.The subcellular localization of MsPrx gene was performed by transient expression vector.The overexpression vector was constructed and the transformation of tobacco by Agrobacterium tumefaciens was used to provide the experimental material for further study.The main results of this experiment are as follows:1.The primers were designed according to the sequence of plant peroxidase gene in Medicago truncatula,and the peroxidase gene MsPrx in alfalfa was obtained by RACE method.Sequence analysis showed that the total length of the gene was 1190 bp,of which CDS was 963 bp,encoding 320 amino acids.Protein sequence analysis showed that the protein has a signal peptide structure,so it is a secretory peroxidase,belonging to the Class III plant peroxidase family.2.The expression of MsPrx gene was found in root,stem and leaf of alfalfa by real-time fluorescence quantitative PCR,but the expression level of the MsPrx gene was the highest in the leaf,followed by the expression in the roots and the least in the stem.The expression of MsPrx in root,stem and leaf increased first and then decreased after NaCl and ABA treatment,indicating that ABA might act as a signal factor to regulate the expression pattern of MsPrx gene under NaCl stress.3.The complete CDS region of MsPrx was cloned by removing the stop codon.The transient expression vector,MsPrx-GFP,was constructed by inserting MsPrx into the pCAMBIA1302-m GFP vector.Subcellular localization was performed by tobacco leaf.The results showed that the MsPrx gene was located in the Golgi complex and may play an important role in plant resistance to stress.4.Construced overexpression vector pCAMBIA1302-MsPrx and transformed into tobacco using Agrobacterium tumefaciens.Identified transgenic tobacco by PCR and providing a reliable experimental material for further research.