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Plant Molecular Biology

, Volume 53, Issue 1–2, pp 213–225 | Cite as

Biochemical evidence linking a putative callose synthase gene with (1→3)-β-d-glucan biosynthesis in barley

  • Jing Li
  • Rachel A. Burton
  • Andrew J. Harvey
  • Maria Hrmova
  • Ahmad Z. Wardak
  • Bruce A. Stone
  • Geoffrey B. Fincher
Article

Abstract

A putative barley (1→3)-β-d-glucan synthase cDNA of 6.1 kb, which is homologous to the yeast FKS gene, was assembled from DNA fragments obtained through screening of barley cDNA and BAC libraries, and by PCR amplification. The corresponding gene, designated HvGSL1, is a member of a family of at least six genes in barley. Gene transcripts are detected at relatively high levels in early developing grain, florets, coleoptiles and roots, but not in leaves infected with a fungal pathogen. A (1→3)-β-d-glucan synthase has been purified more than 60-fold from barley suspension-cultured cells by detergent extraction, CaCl2 treatment, sucrose density gradient centrifugation and non-denaturing gel electrophoresis. The enzyme synthesizes (1→3)-β-d-glucan in vitro and is recognized by antibodies raised against a 17 kDa protein generated by heterologous expression of a fragment of the HvGSL1 cDNA. Furthermore, mass spectrometric analyses show that tryptic peptides produced by in-gel digestion of the active enzyme match peptides predicted from the gene sequence. Thus, the amino acid sequence predicted from the HvGSL1 gene has been linked with the actual amino acid sequence of an active (1→3)-β-d-glucan synthase fraction from barley.

active site gene expression genetic mapping GSL Hordeum vulgare purification 

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Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Jing Li
    • 1
  • Rachel A. Burton
    • 1
  • Andrew J. Harvey
    • 1
  • Maria Hrmova
    • 1
  • Ahmad Z. Wardak
    • 2
  • Bruce A. Stone
    • 2
  • Geoffrey B. Fincher
    • 1
  1. 1.Australian Centre for Plant Functional GenomicsUniversity of Adelaide, Waite CampusGlen OsmondAustralia
  2. 2.Department of BiochemistryLa Trobe UniversityBundooraAustralia

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