biologia plantarum

Biologia plantarum 2015, 59:394-398 | DOI: 10.1007/s10535-015-0485-2

Preparation of vectors with metallothionein gene enriched by additional metal binding domain and their transient expression in Nicotiana tabacum

J. Fišer¹, M. Sura-De Jong^1,2, T. Macek^1,*

¹ Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Czech Republic

² Van Hall Larenstein University of Applied Sciences, Leeuwarden, the Netherlands

The issue of finding plants suitable for phytoremediation of inorganic contaminants can be addressed through the preparation of genetically modified plants with an increased metal accumulation potential. A HisCUP gene, which encodes for a yeast metallothionein fused with a polyhistidine tail (His), was chosen for preparation of two plant vectors. These two plant vectors were constructed and a HisCUP gene expression was subsequently investigated. We firstly prepared a vector pNOV2819/RbcS/HisCUP which enabled selection on a mannose medium and contained the HisCUP gene under an inducible Rubisco promoter. Secondly, we designed a vector pGreen0029/35S/HisCUP which enabled selection of plants on a medium with kanamycin and carried the HisCUP gene under a constitutive CaMV 35S promoter. The transient expression of the HisCUP gene in tobacco plants was confirmed at RNA and protein levels for both constructs. The relative expression of the HisCUP gene was determined by semi-quantitative real-time PCR; a higher expression was detected for the vector pNOV2819/RbcS/HisCUP.

Keywords: heavy metals; HisCUP; phytoremediation; RT-PCR; tobacco

Subjects: metallothioneins; phytoremediation; transgenic plant; binary plasmid; tobacco

Species: Nicotiana tabacum

Received: March 4, 2014; Revised: July 2, 2014; Accepted: August 26, 2014; Published: June 1, 2015Show citation