A CONDITIONAL NEGATIVE SELECTION FOR ARABIDOPSIS EXPRESSING A BACTERIAL CYTOSINE DEAMINASE GENE

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacteria, is usually undetected in higher plants and animal s. 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 fus ed to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 containe d two marker genes for plants: a positive marker gene, bialaphos resis tance (bar) gene driven by the promoter from nopaline synthase gene (P nos) and a negative one, P35S-codA. The binary vector pLABR102 was tra nsformed into Arabidopsis thaliana via Agrobacterium-mediated transfor mation. In transgenic progenies (T3) of the second (T2) generation het erozygous for a single T-DNA insertion, a 3:1 segregation ratio was ob served on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on th e other hand, no segregation was detected: all the T3 seedlings were r esistant 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 Ar abidopsis 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 resist ance are dominant traits in Arabidopsis.