Cloning And Characterization Of A Cytosolic Ascorbate Peroxidase Gene LmAPX From Lycium Chinense Miller

Plant cells produce a large number of reactive oxygen species?ROS? under biotic and abiotic stresses. When plants grow under stress conditions, ROS would be accumulated quickly, and excess ROS can cause damage to plants. Hydrogen peroxide?H₂O₂?, the most stable form among ROS, acts as a precursor of more cytotoxic reactive oxygen derivatives. Soil salinity is one of the most serious environmental stresses. When plants grow under salt stress conditions, large amount of H₂O₂ were produced and severely damage plant growth and metabolism.The ascorbate peroxidase was found in higher plants, eukaryotic algae and some cyanobacteria. As a central enzyme in hydrogen peroxide scavenging, The ascorbate peroxidase can effectively reduce oxidative stress caused by the accumulation of active oxygen in plants.Lycium chinense Mill.?L.chinens? is a solanaceous defoliated shrub which grows in China and other parts of Asia. L.chinense was considered to be a pioneer plant to solve soil salinity in northwest with the high resistance to drought and salt. In this study, a cytosolic ascorbate peroxidase gene was cloned from Lycium chinense Mill.,named LmAPX, using homologous cloning. The main research and result of experiment were showed as following:1. The whole LmAPX cDNA sequence was 965 bp long, consisting of a 753 bp open reading frame and a 212 bp 3'-UTR. The putative LmAPX protein contained250 amino acid residues with an estimated molecular mass of 27.5 kDa. Its deduced amino acid sequence revealed the presence of the conserved amino acid regions of the APX, such as the active sites?H42, W179, D208? and the binding site?H163?.2. The ORF of LmAPX was sub-cloned into prokaryotic expression vector pET28 a. The recombinant protein heterologously expressed in Escherichia coli.showed high activity using l mmol/L IPTG. The recombinant protein was extracted and purified by Ni2+affinity chromatography. The enzyme kinetics constant Km and Vmax were calculated by double-reciprocal plot, and the enzyme activities at differenttemperature and pH were detected.3. The ORF of LmAPX was sub-cloned into yeast expression vector pYES2.Then the recombinant vector was transformed into wild type yeast strain W303. When treated with H₂O₂ and NaCl, the yeast strain harboring LmAPX exhibited better growth compared with the wild type yeast strain.4. The transformation vector pCAMBIA2300-LmAPX was constructed. The recombined plasmid was transferred into Agrobacterium tumefaciense?C58?, and then transferred into wild tobacco with leaf disc transformation. Southern blot analysis indicated that three inserts of LmAPX existed in the tobacco genome. Compared with wild-type tobacco plants, the transgenic plants of over-expressed LmAPX exhibited lower amount of hydrogen peroxide?H₂O₂? and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate?Pn? under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.