基因克隆和表达

Cloning and expression of peroxisomal Ascorbate Peroxidase gene

from wheat

Yaping Chen,Huazhong Wang,Xiue Wang,Aizhong Cao&Peidu Chen*

State Key Laboratory of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University, Nanjing210095,People’s Republic of China;*Author for correspondence(Phone:+86-25-84396026;E-mail: pdchen@)

Accepted24October2005

Key words:peroxisomal ascorbate peroxidase,powdery mildew,SSH,wheat

Abstract

A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase(pAPX)was cloned by Suppression Subtractive Hybridization(SSH)and in silico approach.The cDNA was1027bp in length and contained a complete ORF of876bp,which encodes a protein of292amino acid residues.Its deduced amino acids sequence had84%identity with that of pAPX from barley.The gene was designated as Ta-pAPX.The Ta-pAPX homologous genes were mapped on wheat chromosome7A and7D 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 Yangmai5was enhanced,but its expression in wheat-Haynaldia villosa6VS/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.

Abbreviations:APX–ascorbate peroxidase;ESTs–expressed sequence tags;IPTG–isopropyl-beta-D-thiogalactopyranoside;MW–molecular weight;ORF–open reading frame;pAPX–peroxisomal ascorbate peroxidase;SSH–Suppression Subtractive Hybridization.

Introduction

Ascorbate peroxidase(APX),found in higher plants,cyanobacteria,and algae[1],is the key enzyme in degradation hydrogen peroxide.So far, at leastfive APX isoforms have been identified in plants:cytosolic isoforms,mitochondria isoforms, peroxisomal/glyoxysomal isoform and two chlo-roplastie isoforms,one in stroma and the other associated with the thylakoid membranes,all of which catalyze the reaction:

2ascorbate peroxidaseþH2O2!

2monodehydroascorbateþ2H2O

APXs activity increased in response to a num-ber of stress conditions,such as drought[2],salt [3],high temperature[4]and pathogen infection [5].Relationship between different stress condi-tions and changes of APX activity were observed.

Powdery mildew caused by E.graminis DC.f.sp.tritici is one of the most serious diseases of common wheat in China and many other countries.The Triticum aestivum(‘‘Yangmai5’’)–Haynaldia villosa6VS/6AL translocation line carrying powdery mildew resistance gene Pm:21 confers effective resistance to all current powdery mildew races.To investigate the mechanism of

Molecular Biology Reports(2006)33:207–213

DOI10.1007/s11033-005-4536-1ÓSpringer2006