Transgenic cell lines as a useful tool to study the biochemistry of down-regulation of an endogenous rice gene using a heterologous diamine-oxidase cDNA

Abstract

Transgenic rice (Oryza sativa L.) cell lines engineered with the pea diamine oxidase cDNA in antisense orientation under the control of two different promoters were recovered using particle bombardment. Plasmids p35Sdaoa and pEdaoa contained the pea diamine oxidase cDNA driven by the CaMV35S and the pea ENOD12 nodulin promoter, respectively. Molecular analyses confirmed the stable integration of the transgene and active transcription (mRNA) with both promoters driving the gene of interest. We observed a 2-fold decrease in diamine oxidase activity (P < 0.01) in transformed callus lines expressing p35Sdaoa compared to wild-type or lines expressing the selectable marker gene alone (hpt). In cell lines transformed with pEdaoa, a 2.5-fold reduction in enzyme activity was measured (P < 0.01). Biochemical analysis of cellular polyamines from p35Sdaoa transformants indicated up to a 6-fold increase in putrescine levels (P < 0.001). Concentrations of higher polyamines also increased, with spermidine and spermine levels increasing 8- (P < 0.05) and 3.5-fold (P < 0.01) respectively. A maximum of 2.5-fold increase in putrescine (P < 0.001) and spermidine (P < 0.001) was measured in lines transformed with pEdaoa. No significant variation (P > 0.05) was observed in spermine levels in pEdaoa transformants. We demonstrate for the first time the down-regulation of an endogenous enzyme involved in polyamine metabolism in plants accompanied by a concomitant increase in the concentration of putrescine and spermidine.

Teresa Capell was supported by a grant from the ‘Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria’ (INIA, Spain). The JIC is supported in part from the BBSRC.