Poster: Enzymology
Abs #
247: Molecular cloning and charaterization of b-glucosidases and UDP-Glc glucosyltransferases from Gramineae
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Presenter: |
Sue, Masayuki , sue@nodai.ac.jp |
Authors | Sue, Masayuki (A) Miyamoto, Toru (A) | | Affiliations: |
(A): Department of Applied Biology and Chemistry, Tokyo University of Agriculture
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In some gramineous plants including wheat, rye, and maize, the cyclic hydroxamic acids (Hxs) play an important role in the defense system against pathogens and insects. Hxs are biosynthesized by four P-450s from indole, and UDP-glucose:Hx glucosyltransferase (GT) transfers glucose to Hx to form inactive Hx-glucoside (Hx-Glc). Upon the tissue disruption caused by insect attack or infection of pathogens, b-glucosidase hydrolyzes Hx-Glcs into toxic aglycones and glucose. In wheat, rye, and maize, the concentration of Hx-Glcs and the activity of b-glucosidases and GTs are high in the young plants. To investigate the expression mechanisms and the functions of these enzymes, we isolated the cDNAs encoding glucosidases and GTs from wheat and rye. The full length cDNAs were obtained by screening of cDNA libraries prepared from 48-h-old wheat and rye seedlings. The cDNAs of b-glucosidase precursor of wheat and GTs of wheat and rye were predicted to encode 569-, 456-, and 454-amino acid-long polypeptides, respectively. The wheat b-glucosidase precursor was found to possess a putative N-taminal plastid-targeting transit peptide, similarly to some other known monocot glucosidases. Both b-glucosidase and GT in wheat showed more than 90% identity with the orthologs in rye and 60-70% identity with those in maize. Northern blot analyses showed that mRNAs corresponding to these enzymes are expressed at a high level in young plant shoots. The cDNAs were cloned into an expression vector, and introduced into E. coli cells. After treatment of the transformed cells with IPTG, the expression of proteins was analyzed by SDS-PAGE. The GTs were successfully expressed only when they were constructed as fusion proteins with His-tag at their N-termini.
