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KMID : 0578319930030040363
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Molecules and Cells
1993 Volume.3 No. 4 p.363 ~ p.371
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Isolation and Characterization of an Oat (1-3, 1-4)-β-Glucanase cDNA
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Yun, Song J.
Martin, Debra J./Gengenbach, Burl G./Rines, Howard W./Somers David A.
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Abstract
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Hydrolysis of cell wall (1-3, 14}p-glucan accompanies both the expansion of vegetative cells and the degradation of cereal endosperm cell walls during germination. To investigate the role of (1-3, 14)-p-glucanase [(1-3, 14)-Q-glucan 4-glucanohydrolase; EC 3.2.1.73] in various plant development phases in oat, a 1,448-bp cDNA clone, pOGLI, encoding an (1-3, 14)-(3-glucanase was isolated and characterized. pOGLI contained a short 5´ untranslated region, an open reading frame for 334 amino acids, and a 3´ untranslated region. the amino acid sequence translated from p0GL1 showed more than 90% sequence identity to barley (1-3, 14)-p-glucanases. To confirm that pOGLI coded for a (1-3, 14)-p-glucanase, protein encoded by pOGL1 was produced in Escherichia eofi and purified. The pOGLI-encoded protein had (1-3, 14)-o-glucanase activity and substrate specificity to barley (1-3, 14)-P-glucan, but it did not hydrolyse (1-3)-o-glucan, carboxymethyl-cellulose, or starch. Tri- and tetra-saccharides linked by (1-3)- and (14)-p-linkages appeared to be the major products from action of the pOGLI-encoded enzyme on barley (1-3, 14)-o-glucan. Genomic DNA gel blots probed with pOGLI indicated that oat (1-3, 14)-o-glucanase genes are present at low copy number that may be homoeologous genes from the three different diploid progenitor genomes comprising hexaploid oats. RNA gel blot analyses using the insert of p0GL1 as a probe indicated that steady state levels of (1-3, 14)-a-glucanase-hybridizing transcripts were relatively high in developing oat leaves and in aleurone layers of germinating kernels, intermediate in developing kernels and roots, and low in shoots.
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