Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits
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Heterologous expression of cDNA library 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 a dwarf due to the expression of an ATP synthase, and another vegetative mutant did not produce flowers even after 7 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.
KeywordsFull-length cDNA library Binary vector Plant transformation Tomato Arabidopsis thaliana Heterologous expression
The project was supported by Penn State Erie, The Behrend College. CMM, ASK, MKR, and ADJ were supported in part by Penn State Behrend Undergraduate Student Academic Year Research Grant Program and Undergraduate Student Summer Research Fellowship Program. Daryl J. Nowacki participated in the early phase of the project. We thank Michael A. Campbell and James T. Warren for reviewing the manuscript.
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