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Title: Cloning and characterization of metallothionein genes of Suillus sibiricus an ectomycorrhizal fungus
Authors: Sharma, Anuja
Supervisor: Reddy, M. S.
Keywords: Suillus sibiricus, metallothionein, metal tolerance, functional complementation assays, S. cerevisae mutant strain DTY4 and wild strain BY4741;DBT
Issue Date: 20-Aug-2015
Abstract: Heavy metal pollution is one of the major environmental hazards concerning the world today. Heavy metals are continuously mobilized into our food chain through metal contaminated soil, air and water. Heavy metal contaminated soil hinders the plant growth by entering into the plant cells and altering the cellular metabolism. In such cases mycorrhizal fungi plays a vital role. The mycorrhizal fungi form a symbiotic relationship with the plant roots and protect them from various heavy metal stresses. The ectomycorrhizal fungi have the inbuilt defence mechanism to chelate the heavy metals by producing the metal chelators like, metallothioneins, glutathione, phytochelatins etc. It has been observed that plants associated with the mycorrhizal fungi can sustain high concentrations of heavy metals. Metallothioneins, cysteine-rich polypeptides, are quoted to be responsible for metal detoxification by chelation and homeostasis in eukaryotic organisms. This study focuses on the cloning and characterization of the metallothionein genes, SsMT1 and SsMT2 from Suillus sibiricus into the metal-sensitive yeast mutants to study the expression of the genes in eukaryotic system. The metallothionein genes were firstly identified in the EST library of Suillus species. These EST sequences were characterized using the multiple sequence alignment and phylogenetic analysis. Using these analysis two metallothionein genes were selected and the gene specific primers were designed as SsMTF1, SsMTR1, SsMTF2, SsMTR2. Restriction sites were added to the 5’ end of these primers. The metallothionein genes were amplified using these primers and restriction digestion was performed along with pFL61 using the restriction enzymes EcoRI and BamHI. The ligated product was transformed into E.coli DH5α cells and then into the Saccharomyces cerevisae mutant DTY4 (Copper sensitive strain) which is a mutant of BY4741(Copper tolerant). Expression of SsMT genes was studied by applying yeast functional complementation assay. In this approach the expression of both the genes in response to Cu stress was studied as compared to that of the BY4741 (wild, heavy metal-tolerant) strain. We also studied the level of gene induction under Cu stress at different concentrations of the metal. The mycelium was exposed to different concentrations of CuSO4 and then the biomass was acquired and studied for the effect of different concentrations on the growth of the mycelium.
Description: MS, DBT
Appears in Collections:Masters Theses@DBT

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