tailieunhanh - Báo cáo khoa học: Spatio-temporal profiling and degradation of a-amylase isozymes during barley seed germination

Ten genes from two multigene families encode barleya-amylases. To gain insight into the occurrence and fate of individual isoforms during seed ger-mination, thea-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. | ễFEBS Journal Spatio-temporal profiling and degradation of a-amylase isozymes during barley seed germination Kristian S. Bak-Jensen1 Sabrina Laugesen2 3 Ole Ostergaard1 Christine Finnie1 3 Peter Roepstorff2 and Birte Svensson1 3 1 Carlsberg Laboratory Department of Chemistry Copenhagen Valby Denmark 2 Department of Biochemistry and Molecular Biology University of Southern Denmark Odense M Denmark 3 Enzyme and Protein Chemistry BioCentrum-DTU TechnicalUniversity of Denmark Lyngby Denmark Keywords 2D western blot and mass spectrometry a-amylase isozymes barley seed germination gibberellic acid proteolytic processing Correspondence B. Svensson Fax 45 45 886307 Tel 45 45 252740 E-mail bis@ P. Roepstorff Department of Biochemistry and Molecular Biology University of Southern Denmark Campusvej55 DK-5230 Odense M Denmark Fax 45 65 502467 Tel 45 65 502404 E-mail roe@ These two authors contributed equally to this work Received 16 January 2007 revised 13 March 2007 accepted 15 March 2007 doi Ten genes from two multigene families encode barley a-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination the a-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis western blotting and mass spectrometry. Mass spectrometric analysis confirmed that the 29 a-amylase positive 2D gel spots contained products of one GenBank accession gi 113765 and two gi 4699831 and gi 166985 genes encoding a-amylase 1 and 2 respectively but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both a-amylase 2 entries co-migrated in five full-length and one fragment spot. The a-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass .