Expression of Plant β-Glucosidase Genes in Schizosaccharomyces Pombe

Abstract

ABSTRACTSchizosaccharomyces pombe as a model host organism was used for the functional analysis of plant genes. The heterologous expressions of dhrl and myrl genes encoding β-glucosidase in Sorghum bicolor and Sinapis alba, respectively, have been investigated in this fission yeast. cDNA's of these genes are cloned in pART1 and pREP42 expression vectors and transferred to S. pombe leu1–32 and ura4-D18 strains by electroporation. The transformants are screened by using colony PCR to identify the colonies containing plant genes. It was determined that pART1 was not a suitable vector for cloning these genes. Therefore, further analyses were done using recombinant vectors derived from pREP42. Transcripts of the genes from the recombinant clones were detected by RT-PCR using gene specific primers. Crude protein extracts prepared from these clones were analyzed by SDS-PAGE and Western blotting. In addition, these extracts were assayed for the enzyme activity. It was determined that plant β-glucosidase genes were expressed at transcriptional and translational levels in S. pombe, but their protein products were inactive. These results may indicate some deficiencies in the post translational modifications or negative interactions between S. pombe and plant β-glucosidases.