Cloning and Characterization of Metallothionein Genes of Ectomycorrhizal Fungi Suillus himalayensis and Suillus indicus

Abstract

Adverse effects of heavy metals have been known for a long time and exposure to heavy metals continues and is even rapidly increasing. Use of mycorrhizal fungi is one of the best known methods to cope up with heavy metal contamination. Mycorrhizal fungi plays a vital role to ensure plant survival and establishment of vegetation in heavily polluted places as it is in direct contact with plants and soils. In this study we have focused on molecular mechanisms in metal detoxification processes. In this study, metallothioneins were identified from, ectomycorrhizal fungi, Suillus himalayensis and Suillus indicus to obtain a deeper insight into the detoxification mechanisms of toxic heavy metals by the organism. These two strains were checked for their heavy metal tolerance on copper by growing them on liquid medium supplemented with different concentrations of copper. In result, we found that there was significant decrease in growth by increasing concentration of Cu. Primers were designed by retrieving the EST library from the genome of Suillus luteus. Five different genes encoding MTs were identified in the genome of Suillus luteus. For MT gene induction, mycelium was exposed to heavy metal stress. RNA was isolated from the mycelium and cDNA was synthesized. cDNA was amplified wth gene specific primers to verify cDNA. Four metallothionein genes were recognized and further cloned and characterized. Metallothioneins genes were screened for metal resistance in Eukaryotes by transformation of metal-sensitive Saccharomyces cerevisae mutants. To validate the functionality of the genes, Yeast functional complementation assays were performed. Genes were expressed in one Saccharomyces cerevisae mutant strain, and the transformants were monitored on synthetic media, SD-Ura, with and without metal supplements. The present study identifies metallothionein from Suillus indicus and Suillus himalayensis as a potential tool against copper.