Molecular cloning & In silico Analysis of Metallothionein Type II Gene from Leucaena leucocephala (a Leguminous Species)

Plant metallothioneins (MTs) are low molecular weight cysteine rich proteins consisting of a single polypeptide chain with two thiol-rich sites - α and β domain which have a strong affinity to metal ions. Apart from heavy metal detoxification, plants MTs are involved in regulation of essential metals and have been shown to be responsive toward other abiotic stresses. Some plants have evolved physiological and molecular mechanisms like compartmentalisation, sequestration, chelation or exclusion against metal stress. Leucaena leucocephala, a leguminous tree species has heavy metal tolerance and survival ability in inhospitable conditions. In this study, Metallothionein type II (LlMTII) gene was isolated from L. leucocephala and cloned. Sequencing revealed the Open Reading Frame of 228 bp in length which encoded putative protein composed of 75 amino acids with a molecular mass of 7.671 kDa and theoretical pI of 4.90. Negative GRAVY number indicated hydrophilic nature of the protein, aliphatic and instability indices showed unstable nature in in-vitro conditions. LlMTIIbelonged to p1 sub-family of Metallothioneins and 3D modeling showed two Cys-rich domains with a long Cys-free linker of about 44 aa. It exhibited association with various abiotic stress responsive transcription factors. Multiple sequence alignment and phylogenetic analysis showed high level of similarity in the conserved domains especially Cys residues with many species (both Monocots and Dicots). This study helps us to better understand LlMTII gene and its role in heavy metal detoxification and conferring tolerance to other abiotic stresses.