Abstract
Gene targeting (GT) by homologous recombination is the most powerful technique available for specific modification of a chromosomal target gene. In angiosperms, the transferred DNA (T-DNA) is randomly integrated into the genome by non-homologous end-joining (NHEJ)-related mechanism, therefore GT efficiency is extremely low. Thus, it is expected that suppression of NHEJ pathway increase GT frequency. It has been recently reported that the lack of AtLigIV cause an increase in the efficiency of GT.
We generated rice plants (KD-Ku70, Ku80 and LigIV) transformed with an RNAi construct using 3' sequence of Ku 70, Ku80 and LigIV mRNA. To test the efficiency of T-DNA integration, KD calli were infected with Agrobacterium harboring reporter genes ; 35S pro+GFP and Eluc gene trap constructs. 3-day after infection, the transient expression of GFP in KD calli were comparable to that in WT callus. 9-day after infection, LUC signals, monitoring stable expression of the trans-gene, were suppressed in KD plants compared to control plants. We will investigate the effect of the suppression of NHEJ on the efficiency of HR using HR repair assay system.
