Plant Genome IV Conference Plant Genome IV Conference
Random cDNA sequencing is an efficient way of isolating expressed plant genes massively. However, functional identification of the cDNA clones in this approach is limited to the clones with similarities to known genes. This approach, thus, may not identify novel genes functioning in plants. To overcome this limitation, we are examining a new approach, which we call random antisense mutagenesis (RAM).
We have constructed an antisense cDNA library in Agrobacterium with an equal amount of poly (A)+ RNA extracted from each major organ of Arabidopsis. This library was used to generate transgenic tobacco plants en masse. Visual screening of the transgenic tobacco plants revealed that 10% of the transgenic plants have mutant phenotypes. The mutant phenotypes include elongated hypocotyl, early senescence, dwarfism, defective shoot meristem, pointed cotyledon, etc. Most of these mutations are segregating as single dominant characters in the T2 generation, as expected for mutations caused by antisense blocking of gene expression. Furthermore, three mutant lines we examined showed cosegregation of the mutant phenotypes with the inserted T-DNA.
These data suggest that RAM may be a powerful approach to isolate novel plant genes with known functions at a relatively large scale with a reasonable amount of effort.