Cloning and expression of peroxisomal ascorbate peroxidase gene from wheat.
A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase (pAPX) was cloned by Suppression Subtractive Hybridization (SSH) and in silico approach. The cDNA was 1027 bp in length and contained a complete ORF of 876 bp, which encodes a protein of 292 amino acid residues. Its deduced amino acids sequence had 84% identity with that of pAPX from barley. The gene was designated as Ta-pAPX. The Ta-pAPX homologous genes were mapped on wheat chromosome 7A and 7D using Chinese Spring nulli-tetrasomic lines analysis. Northern analysis indicated that, after inoculation by Erysiphe graminis Dc.f.sp. tritici, the expression of Ta-pAPX gene in Yangmai5 was enhanced, but its expression in wheat-Haynaldia villosa 6VS/6AL translocation lines changed a little. The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew. The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32 (a+) and a protein with the same deduced molecular weight (MW) was expressed in E. coli BL21 (DE3), which showed ascorbate peroxidase activity.
Chen
Yaping
Y
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
Wang
Huazhong
H
Wang
Xiue
X
Cao
Aizhong
A
Chen
Peidu
P
D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D001483:Base Sequence; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics*; D020675:Peroxisomes / Q000201:enzymology*; D016415:Sequence Alignment; D014908:Triticum / Q000201:enzymology* / Q000235:genetics*
D010544:Peroxidases; D060387:Ascorbate Peroxidases