Cloning Of MsPOD Gene From Alfalfa And Transformation Of Arabidopsis Thaliana

In this study, the full length of peroxidase gene, MsPOD was obtained from alfalfa "Zhongmu No.1" by homologous cloning technology, and the plant expression vector pBI-POD was constructed. Transgenic Arabidopsis plants were obtained by floral dip method with Agrobacterium tumefaciens GV3101harboring pBI-POD plasmid. This work will help characterize the function of MsPOD gene and improve plants by transgene with this gene. The results of this study as follows:1、 MsPOD was cloned from alfalfa by RT-PCR technology, the bioinformatics analysis of MsPOD showed that the full-length open reading fragment was1062-bp in length, encoding353amino acid residues. Via the gene homology and phylogenetic tree analysis, we can see that MsPOD gene was high homology compared with other plant peroxidase gene and also prove they all belongs to the class III peroxidase multigenic family, meanwhile, the MsPOD gene is a member of the secretory peroxidase.2、Real-time RT-PCR was performed to reveal transcript level of MsPOD in different tissues and under different abiotic stresses. The results indicated MsPOD was abundant in stem, leaf and flower, rather than in root; the results showed that the expression of MsPOD in different processing time has more significant difference, and in different stress conditions, MsPOD gene expression has its unique, especially in the treatment conditions of H2O2and NaCl, gene expression have a more significant increase in volume. Thus MsPOD under common gene induced H2O2and salt.3、To further investigate function of this gene in plants, The coding damain sequence of MsPOD was inserted into the pBI121, constructed a plant expression vector, which was introduced into Arabidopsis plants by Agrobacterium tumefaciens. Thirteen Arabidopsis plants with kanamycin resistance were obtained. Three Arabidopsis plants have been confirmed as positive transgenic plants by PCR and RT-PCR. Histochemical staining showed that GUS active appeared in inflorescences, which further confirmed that MsPOD gene was successful expressed in Arabidopsis thaliana.4、The fusion was then transformed into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed MsPOD-GFP fusion was detected by a confocal laser scanning microscope. The result revealed that MsPOD localized in nucleus.