title: Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli.
abstract: Salicornia brachiata is an extreme halophyte growing luxuriantly in the coastal marshes and frequently exposed to various abiotic stresses including heavy metals. A full length type 2 metallothionein (SbMT-2) gene was isolated using RACE and its copy number was confirmed by southern blot analysis. Transcript expression of SbMT-2 gene was analyzed by semi-quantitative Rt-PCR and real time quantitative (qRT) PCR. Expression of SbMT-2 gene was up-regulated concurrently with zinc, copper, salt, heat and drought stress, down regulated by cold stress while unaffected under cadmium stress. Heterologous expression of SbMT-2 gene enhances metal accumulation and tolerance in E. coli. Metal-binding characteristics of SbMT-2 protein show its possible role in homeostasis and/or detoxification of heavy metals. Significant tolerance was observed by E. coli cells expressing recombinant SbMT-2 for Zn(++), Cu(++) and Cd(++) compared to cells expressing GST only. Sequestration of zinc was 4-fold higher compared to copper and in contrast SbMT-2 inhibits the relative accumulation of cadmium by 1.23-fold compared to GST protein. Fusion protein SbMT-2 showed utmost affinity to zinc (approx. 2.5 fold to Cu(++) and Cd(++)) followed by copper and cadmium ions with same affinity. Halophyte S. brachiata has inherent resilience of varying abiotic tolerance therefore SbMT-2 gene could be a potential candidate to be used for enhanced metal tolerance and heavy metal phytoremediation.
authors.affiliation: Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research, GB Marg, Bhavnagar-364002, Gujarat, India.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Chaturvedi
authors.forename: Avinash
authors.initials: A
authors.lastname: Mishra
authors.forename: Vivekanand
authors.initials: V
authors.lastname: Tiwari
authors.forename: Bhavanath
authors.initials: B
authors.lastname: Jha
mesh_terms: D000595:Amino Acid Sequence; D019663:Chenopodiaceae / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055051:Salt-Tolerant Plants / Q000235:genetics
chemical_list: D010940:Plant Proteins; D008668:Metallothionein