A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene.

Abstract

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals. The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth. The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA. The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC. PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome. The results indicated that cytosine deaminase from E. coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.