T. Kobayashi et al., A CONDITIONAL NEGATIVE SELECTION FOR ARABIDOPSIS EXPRESSING A BACTERIAL CYTOSINE DEAMINASE GENE, Idengaku Zasshi, 70(3), 1995, pp. 409-422
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.