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Elimination of Hindm by site-directed mutagenesis facilitated the cloning of
cah gene and construction of plant transformation vector pCAMBIA-cah. The wild
type cah gene and the mutated cah genes devoid of HirtdI11 were successfully cloned
and overexpressed in E. coli and the purified enzymes from these genes showed
similar level of cyanamide hydratase activity. Results revcaled that single base site-
mutation does not have effect in the activity of the protein. The cyanamide hydratase
(244
amino acid) encoded by cuh gene converts cyanamide, a compound high in
nitrogen that is toxic to plants cells into urea which is non-toxic, commonly used
fertilizer. The use of cah gene as a marker is desirable since the most co~nmonly used
selectable markers are antibiotic and herbicide resistance genes that has caused public
concern about the possible transfer of these genes to unintended species such
as
microbial pathogens and weeds (Mike and Mc Hugh, 2004).
For the first time, present study described the expression of mutated cah gene
devoid of Hind111 in tobacco plants. Agrohacterium method was found to be efficient
biological method of gene transfer. The non-transformants did not form roots,
exhibited yellow necrosis and died due to cyanamide toxicity. Colorimetric assay for
decrease in cyanamide levels upon incubation with plant extracts provided an efficient
means to verify the putative transformants expressing cyanamide hydratase. This
assay is found to be a rapid and easy method to screen putative transformants.
Molecular analyses of transformants by PCR and Southern blot analyses
further confirmed the integration of cah, chi and Ltp. It is now known that multiple
gene copies frequently lead to co-suppression and gene silencing (Vaucheret et al.,
1998). Transgene copy number can be positively or negatively associated with
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