Cloning and characterization of cottonGhBGgene encoding β-glucosidase

Abstract

Beta-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding beta-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced.