Cloning of gene encoding ß-glucosidase enzyme from olive (Olea europaea) leaves

ß-glucosidases that belong to the family 1 glycoside hydrolyses catalyze the hydrolysis of the ß-glucosidic bond. In plants ß-glucosidases play important roles in diverse aspects of plant physiology including formation of intermediates in cell wall lignification, activation of phytohormones and activation of metabolites related to chemical defense.In a previous study in our laboratory, a cDNA library was constructed which containing 2.4X106 independent clones from leaves of olive (Olea europaea L.) and contigs and singlets were generated using both Phrap and CAP3 bioinformatic programmes. The candidate genes were searched which can be amenable for biotechnological applications and was identified a contig sequence that shows homology to genes encoding ß-glucosidases in plants. The aim of this study is to isolate and clone full length ß-glucosidase cDNA from leaves of olive.Expression of genes encoding ß-glucosidase homologs are differentially upregulated in response to cold therefore total RNA isolated from olive leaves of exposed to cold stress. Forward and reverse primers were designed using sequence information of the contig. RT-PCR reactions were performed to clone full lenght cDNA and a fragment about 1700 bp was obtained. The fragment was cloned and sequenced. The sequence showed more than 98% similarity to olive beta glucosidase in BLASTN and BLASTP analysis.In later stages of an ongoing research project, full length cDNA was obtained coding region will be inserted into Pichia expression vector and the protein will be expressed in Pichia pastoris.