Expression and characterization analysis of type 2 metallothionein from grey mangrove species (Avicennia marina) in response to metal stress.
Metallothioneins (MTs) are a family of low-molecular-weight cysteine-rich proteins and are thought to play possible roles in metal metabolism or detoxification. To evaluate the roles of metallothioneins in metal homeostasis or tolerance in Avicennia marina, a real-time quantitative PCR protocol was developed to directly evaluate the expression of AmMT2 mRNA, when A. marina seedlings were exposed to different concentrations of zinc (Zn), copper (Cu) or lead (Pb) for 3 and 7d. Real-time quantitative PCR results indicated that the regulation of AmMT2 mRNA expression by Zn, Cu and Pb was strongly dependent on concentration and time of exposure. A significant increase in the transcripts of AmMT2 gene was also found in response to Zn, Cu and Pb, at least under some experimental conditions. When AmMT2 was overexpressed in Escherichia coli BL21 as a carboxy-terminal extension of glutathione-S-transferase (GST), the transgenic bacteria showed an increased tolerance to Zn, Cu, Pb and Cd exposure as compared to control strains. Moreover, GST-AmMT2 was purified from E. coli cells grown in the presence of 400 microM Zn, Cu, Pb or Cd. The purified GST-AmMT2 fusion protein could bind higher levels of all four metals than GST alone. Taken together, these data support the hypothesis that AmMT2 may be involved in processes of metal homeostasis or tolerance in A. marina.
Huang
Guo-Yong
GY
Key Laboratory of Tropical Marine Environmental Dynamics, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. huang gyh@sina.com
Wang
You-Shao
YS
D032407:Avicennia / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002104:Cadmium / Q000633:toxicity; D003300:Copper / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D005982:Glutathione Transferase / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D007854:Lead / Q000633:toxicity; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015032:Zinc / Q000633:toxicity
D008670:Metals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014874:Water Pollutants, Chemical; D002104:Cadmium; D007854:Lead; D003300:Copper; D008668:Metallothionein; D005982:Glutathione Transferase; D015032:Zinc