Cloning and characterization of dicer like protein from the ectomycorrhizal fungus Laccaria bicolor

Abstract

Laccaria bicolor which is an ectomycorrhizal fungus was used for the present study and the aim was to clone and characterize dicer like protein from it. The sequence of dicer gene was obtained from the Laccaria bicolor genome database. Various bioinformatics’ tools were utilized for the analysis of conserved regions, domains throughout the history of evolution. Phylogenetic trees were constructed and their analysis proved that the dicer like proteins are present in various species of plants, mammals, insects and fungi. Laccaria bicolor was grown on MMN media at 25°C. RNA isolation from the fungus was done and the cDNA was prepared. Primers designed for the gene were used for amplification. The expression vector pET23a was used for the study, it was digested with respective enzymes whose sites were introduced in the primers. The gene was also digested with the same enzymes and further ligated with pET23a in order to proceed with transformation experiments where this recombinant plasmid was put into the E.coli DH5ɑ cells and further into the E.coli BL21De3 cells. Total protein isolation was done to confirm that protein production takes place via IPTG induction. Laccaria bicolor was grown on various nitrogen sources, providing stress and the growth of the fungus was monitored, both in terms of diameter and dry weight. Different concentrations of carbon source (glucose) were also used to monitor the growth pattern of the fungus. Expression of dicer gene in the presence of different glucose concentrations was tested and the level of expression variation was monitored.