Production and characterization of a large population of cDNA-overexpressing transgenic rice plants using Gateway-based full-length cDNA expression libraries

Abstract

We applied the full-length cDNA overexpressor (FOX) gene-hunting system for systematic and genome-wide functional analysis of rice genes. In this study, we constructed a novel binary vector carrying the Gateway site-specific recombination cassette and then constructed rice FOX libraries containing a maximum of 13,823 independent, full-length cDNAs (fl-cDNAs) that correspond to approximately half the total number of rice fl-cDNA clones. By introducing the FOX libraries via Agrobacterium, we generated 2,586 FOX-rice lines exhibiting various visible alterations (e.g., plant height, tillers, leaves, and heading dates). The introduced flcDNAs, integrated into individual transgenic rice genomes, were amplified by genomic PCR and identified using sequencing analysis. The fl-cDNAs were PCR-amplified in 2,251 (94.2%) of the 2,389 FOX-rice lines that were examined, identifying 1,920 independent fl-cDNAs in the FOX lines. In addition to the previously generated FOX-rice plants, our new collection of FOX-rice lines produced through the Gateway system should be a useful tool for the efficient identification of gene functions in rice. Moreover, this Gateway-based technology should be applicable to other species in which a collection of fl-cDNA clones is available.