Cloning and expression analysis of a blue copperbinding protein gene from Dasypyrum Villosum

Abstract

Adifferentially expressed fragment EST145 was isolated by suppression subtractive hybridization (SSH) method. Using EST145 as the probe, a blue copper-binding protein gene designated as DvBCB was screened from Dasypyrum villosum cDNA Library. The DvBCB gene was 845 bp in length with an open reading frame (ORF) which encoded a 178-amino acid polypeptide and contained the deduced functional sites: H₆₆, C₁₀₇, H₁₁₂ and M₁₂₁. Northern blot analysis showed that, the expression of DvBCB gene was enhanced in leaves after inoculation with Erysiphe graminis; reached a peak level at 24 h and decreased to constitutive level at 72 h after inoculation in resistant Gh21 line. The expression level in susceptible mutant M14S line was slightly lower than that in the resistant Gh21 line at all stages after inoculation, and the peak could not appear in M14S line. The function of DvBCB gene might include lignification of cell wall or scavenging of reactive oxygen species (ROS) during powdery mildew attack.

Key words: Dasypyrum villosum, powdery mildew, suppression subtractive hybridization, blue copper-binding protein gene.