Cloning, expression and purification of an ascorbate peroxidase gene from Rhus chinensis

摘要

The sumac species Rhus chinensis is widely used in traditional Chinese medicine. APX is the key enzyme in hydrogen peroxide degradation, and may have a critical function in plant-aphid interactions. Both the cDNA and genomic sequences encoding the APX protein in R. chinensis (termed RcAPX) underwent cloning. The 1938bp RcAPX gene encompassed 6 introns and 7 exons. The open reading frame of RcAPX was 750bp and encoded a 249-amino acid peptide. Based on sequence alignment and phylogenetic analysis, RcAPX was shown to be cytosolic. Sequence alignment revealed that RcAPX shared 54.1-75.7% identity with APX sequences reported in other plant species. Quantitative real-time PCR showed differential expression of RcAPX mRNA in different tissues at various developmental stages. Moreover, RcAPX expression was significantly suppressed by galls. To further assess its function, RcAPX was produced in E. coli using the pET-28a plasmid, and the recombinant peptide showed high APX activity. The optimum temperature and pH for RcAPX activity were 60 degrees C and 8.5. To confirm the substrate binding sites and active sites of RcAPX, site-directed mutagenesis studies were performed. The present study firstly described a full-length APX gene in the family Anacardiaceae.