Site-Directed Mutagenesis, Heterologous Expression of Cyanamide Hydratase Gene and Antimicrobial Activity of Cyanamide

Abstract

Site-directed mutagenesis on a recombinant plasmid, pUC8, that contained the cah gene, was conducted and confirmed by sequence analysis. Single base substitution, G to A at nucleotide position 81 or T to C at nucleotide position 84 of cah gene does not change the amino acid sequence of cah enzyme but eliminates the HindIII site. The wild-type cah and its mutants were cloned and overexpressed in pQE-60 Escherichia coli expression system. Western blot analysis confirmed the production of 27.7-kDa cah enzyme by all the recombinants. The mutated cah gene devoid of HindIII site was used to generate a recombinant plant transformation vector (pCAMBIA-cah). Agrobacterium-mediated transformation was performed in Nicotiana tabaccum cv. Samsun plants by employing the leaf-disc method. The integration and expression of cah gene in transgenic plants were confirmed by polymerase chain reaction, Southern and Western blot analyses. Antimicrobial activity of cyanamide against phytopathogenic fungi and bacteria was determined. Cyanamide can be used as fertilizer as well as an antimicrobial salt against phytopathogenic fungi and bacteria. The present investigation reports the heterologous expression of the cah marker gene. Due to its innate ability to convert cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can be used to facilitate plant growth promotion and biocontrol of phytopathogens.

The genome of newly classified Ochroconis mirabilis: Insights into fungal adaptation to different living conditions

“…It is used as a nitrogen fertiliser in the form of calcium salt and hydrogen cyanamide. Cyanamide was also reported to be phytotoxic, bactericidal and fungicidal [ 19 , 20 ]. In this genome, two genes were identified to encode cyanamide hydratase (UM578_11171) and urease (UM578_4352), respectively and are shown to be expressed (Additional file 2 : Figure S5).…”

“…Cyanamide hydratase catalyses the hydrolysis of cyanamide to urea while urease converts the urea to ammonium. The cyanamide hydratase from the fungus Myrothecium verrucaria is well characterised and used in transgenic plants to act as a biocontrol of phytopathogens [ 20 ]. Moreover, the end product, urea, is a useful compound that acts as a plant fertiliser to facilitate plant growth [ 20 ].…”

“…The cyanamide hydratase from the fungus Myrothecium verrucaria is well characterised and used in transgenic plants to act as a biocontrol of phytopathogens [ 20 ]. Moreover, the end product, urea, is a useful compound that acts as a plant fertiliser to facilitate plant growth [ 20 ]. The presence of genes involved in biodegradation shows the adaptation of the fungus towards the occurrence of non-natural compounds in the environment.…”

Advances in selectable marker genes for plant transformation

“…The enzyme has extremely narrow substrate specificity. The use of cyanamide hydratase as a selectable marker has been demonstrated in wheat (Weeks et al, 2000), tobacco (Maier-Greiner et al, 1991b;Kirubakaran and Sakthivel, 2008), potato, tomato, rice, Arabidopsis (Damm, 1998) and soybean (Zhang et al, 2005;Ulanov and Widholm, 2007). In a recent study, the heterologous expression of cah marker gene was reported.…”

“…In a recent study, the heterologous expression of cah marker gene was reported. Due to its innate ability to convert calcium cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can also be used to facilitate plant growth promotion and biocontrol of phytopathogens (Kirubakaran and Sakthivel, 2008).…”