Cloning and characterization of metallothionein gene LbMT3 isolated from ectomycorrhizal fungus Laccaria bicolor

Abstract

Heavy metals are the main source of soil contamination which effects the growth and development of living organisms. To tackle this problem a large number of remediating processes are available out of them the use of ectomycorrhizal fungi for detoxifying the heavy metals, is the most reliable method. They enhance the plant growth and wellness in severely contaminated soils against metal toxicity. The ectomycorrhizal fungi follow many molecular mechanisms to avoid the uptake or to tolerate the heavy metal stress. In the current study, Laccaria bicolor (ectomycorrhizal fungus) was studied for metal tolerance at different metal concentrations. In many eukaryotes, the cysteine rich peptides i.e. metallothioneins involved in metal tolerance and metal homeostasis are present. Total six putative metallothionein genes of Laccaria bicolor have been reported till date and two genes (LbMT1 and LbMT2) out of them have already been studied. In the present study LbMT3 gene (35 aa long) from Laccaria bicolor was cloned and characterized under metal stress conditions. In the metal tolerance studies, a significant decrease in the growth of mycelium was observed with the increase in metal concentration and from the experimental data LD50 value was also calculated for copper and cadmium for Laccaria bicolor. The gene expression of LbMT3 was studied with the help of qRT-PCR after isolating the RNA and synthesis of cDNA from it. LbMT3 gene showed different expression patterns to different metal stresses. The maximum expression was observed at the highest concentration of metals (40 μM for Cd and 400 μM for Cu). Yeast complementation studies in metal sensitive yeast strains indicated that LbMT3 gene is resistant to both the metals (cadmium and copper) but LbMT3 gene showed significantly higher expression levels in cadmium as compared with copper. This study proves to be highly useful in understanding the different mechanisms involved metal detoxification in fungus which can be further used as potential tool against cadmium and copper stress can be used in bioremediation.