caa a22 4500 463160118 CHVBK 20180406164745.0 cr unu---uuuuu 170326e20070901xx s 000 0 eng 10.1007/s10535-007-0096-7 doi (NATIONALLICENCE)springer-10.1007/s10535-007-0096-7 cDNA cloning, expression, protein purification, and characterization of a novel glyoxal oxidase related gene from Vitis pseudoreticulata [Elektronische Daten] [B. Zhou, X. Wang, Y. Wang] 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. Institute of Experimental Botany, ASCR, 2007 Chinese wild Vitis species nationallicence fusion protein expression nationallicence gene cloning nationallicence mRNA differential display nationallicence polyclonal antibodies nationallicence GLOXrg : glyoxaloxidase related gene nationallicence GLOX : glyoxaloxidase nationallicence GST : glutathione S-transferase nationallicence IPTG : isopropyl-β-D-thiogalactoside nationallicence DDRT-PCR : differential display reverse transcription-PCR nationallicence Zhou B. Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut Wang X. Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut Wang Y. Key Laboratory of Northwest Horticulture, Plant Germplasm and Genetic Improvement of Ministry of Agriculture, College of Horticulture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut Biologia Plantarum Kluwer Academic Publishers-Consultants Bureau 51/3(2007-09-01), 458-466 0006-3134 51:3<458 2007 51 10535 https://doi.org/10.1007/s10535-007-0096-7 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10535-007-0096-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zhou B. Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut NATIONALLICENCE 700 1- Wang X. Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut NATIONALLICENCE 700 1- Wang Y. Key Laboratory of Northwest Horticulture, Plant Germplasm and Genetic Improvement of Ministry of Agriculture, College of Horticulture, Northwest A&F University, 712100, Yangling Shaanxi, P.R. China aut NATIONALLICENCE 773 0- Biologia Plantarum Kluwer Academic Publishers-Consultants Bureau 51/3(2007-09-01), 458-466 0006-3134 51:3<458 2007 51 10535 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer