Cloning and sequencing of a cDNA for poplar endo-1,4-beta-glucanase

Abstract

A cDNA for poplar endo-1,4-beta-glucanase was cloned by use of a synthetic oligonucleotide as probe. The probe was designed on the basis of the N-terminal amino acid sequence of the beta-glucanase from suspension-cultured poplar cells (Populus alba L.), and by complete nucleotide sequence of the cDNA was determined. The 1,614-bp cDNA contained an open reading frame of 1,482 base pairs, encoding 494 amino acids. Removal of a putative signal sequence from the deduced amino acid sequence of the polypeptide yielded a mature protein of 467 amino acids. Comparison of deduced amino acid sequences revealed that the poplar endo-1,4-beta-glucanase was 80% and 70% identity to avocado fruit and bean abscission endo-1,4-beta-glucanases, respectively. Hydropathy plot analysis of the deduced amino acid sequence suggests that poplar endo-1,4-beta-glucanase belongs to family E in terms of the cellulase catalytic domain, and avocado fruit and abscission bean endo-1,4-beta-glucanases also belong to this family. 2,4-D markedly increased the level of the endo-1,4-beta-glucanase mRNA in cultured cells, while GA3, benzyladenine and abscisic acid each repressed transcription of this mRNA. The transcript was also detected in the roots and stems of intact plants, although the level of mRNA was much lower in intact tissues than in cultured cells. Genomic Southern analysis indicated that a small family of gene for endo-1,4-beta-glucanase exists in poplar.