Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli
- 作者：Amit Kumar Chaturvedi ; Avinash Mishra ; avinash@csmcri.org ; Vivekanand Tiwari ; Bhavanath Jha ; bjha@csmcri.org
- 关键词：Abiotic stress ; GST fusion protein ; Heavy metals ; Metal binding ; Metal toxicity ; Phytoremediation
- 刊名：Gene
- 出版年：2012
- 期刊代码：73_03781119
- 类别：bio
- 出版时间：15 May, 2012
- 卷：499
- 期：2
- 页码：280-287
- 文件大小：947 K
摘要
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.