CDNA cloning, expression, protein purification, and characterization of a novel glyoxal oxidase related gene from Vitis pseudoreticulata

Abstract

To study the defense mechanism of the resistance to the disease of Chinese wild Vitis species and offer powerful bases for the molecular breeding of highly disease-resistant grape cultivars, using mRNA differential display reverse transcript-PCR (DDRT-PCR) and RACE, a full-length cDNA was isolated from Chinese wild Vitis pseudoreticulata clone Baihe-35-1 inoculated with Uncinula necator by pressing infected leaves under natural field conditions. The cDNA designated as GLOXrg is 1708 bp in length with an open reading frame of 1572 bp encoding 523 amino acids, containing the conserved domain, glyoxaloxid N domain (pfam07250). No homologous nucleotide sequence was found, but the deduced amino acid sequence of GLOXrg shows 74 % identity with a putative protein from Arabidopsis thaliana (accession No. CAB88357). GLOXrg was cloned into pGEX-4T-1 vector. The recombinant vector expressed an about 83-kDa GST-GLOXrg fusion protein as insoluble inclusion bodies in Escherichia coli BL21. Fusion protein GST-GLOXrg was isolated and used to raise the polyclonal anti-(GST-GLOXrg) in rabbits. Western blot analysis showed a high titer (10 000) and the high specificity of the polyclonal anti-(GST-GLOXrg). This is the first report on the cloning of the full-length GLOXrg cDNA from the plant except Arabidopsis thaliana. Through the differential expression, GLOXrg probably plays a crucial role in the resistance to Uncinula necator of Chinese wild Vitis species. An optimized system on prokaryotic expression, protein purification and preparation of the polyclonal antibodies of GLOXrg was also established.