PREPARATION OF SUPER-TRANSGENIC PLANTS EXPRESSING BOTH DIOXYGENASE AND METALLOTHIONEIN GENES

The aim of this work is the preparation of transgenic plant resistant to both organic and inorganic pollutants. First transgenic plants containing bacterial bphC gene were prepared. The bphC gene is coding for 2,3dihydroxybiphenyl- 1,2-dioxygenase, cleaving the biphenyl ring to form a product which could be further metabolized. The presence of bphC gene in first and second filial generation of transgenic plants was detected on the DNA and RNA level using PCR or RT-PCR methods. Transgenic plants contain the bphC gene in fusion with genes for detection markers GUS (gene for beta-glucuronidase) and LUC (gene for luciferase) and also bphC gene with histidine tail. The expression of the bphC/GUS and the bphC/LUC genes in plants was tested by histochemical assay proving the expression of detection markers. Also the expression of the bphC/His gene was verified after purification of the enzyme by affinity chromatography followed by Western blot and immunochemical assay. The expression level differed among transgenic lines. Based on results of degradation experiment with 2,3-dihydroxybiphenyl, the transgenic lines G1, H1 and L1 were transformed by plasmid carrying yeast metallothionein gene CUP in fusion with histidine tail. The metallothioneins are known for their ability to bind heavy metals, histidine tail is an additional binding domain enhancing the effect. The transformation of transgenic plants was mediated by Agrobacterium tumefaciens. Transgenic plants containing both bphC and His/CUP genes were tested and the transcription of genes was confirmed using transient expression as well as permanent transformation.