Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/1/2009
Publication Date: 12/18/2009
Citation: Wang, Y., Mosebach, C.M., Kibbe, A.S., Ryhal, M.K., Jones, A.D., Palmer, J.A., Kochian, L.V. 2009. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits. Plant Molecular Biology Reporter. 27:454-461. Interpretive Summary: Challenge in functional genomics is to identify functions of all the genes in a genome. One way to do this is to over-express random genes (cDNAs) from a cDNA library in transgenic plants. Dominant mutations found in the resulting transgenic plants may help elucidate gene function. In this paper, we use Arabidopsis as a platform to express cDNAs from tomato fruits to demonstrate that heterologous expression can be used to functionally identify genes from crop plants. The system described in this paper can be used to functionally characterize genes from crop plants for which well-characterized cDNA collections may not be available. Because full-length genes are not available in most plant species, the system described here is useful for investigators studying these plant species to conduct similar gene identification experiments and subsequent functional analysis. In this paper the library has been used for heterologous expression in Arabidopsis to generate new phenotypes to improve agricultural plants. Moreover, the library could also be used to transform any other plants for which an efficient genetic transformation system is available. Therefore, this system could be a very powerful tool for functional genomics studies.
Technical Abstract: Heterologous expression of cDNA libraries in Arabidopsis and other plants has been used for gene identifications. To identify functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis, with cDNA sizes of 2-5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that 75% (61) are full-length genes which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform Arabidopsis plants. Among the 7,000 mutants, one was found to be dwarf due to the expression of an ATP synthase and another vegetative mutant did not produce flowers even after seven months. The technique was validated by reintroducing the tomato ribosomal protein L9 gene and can be used in any other plant species as a gene discovery tool.