Date of Graduation

12-2015

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Andy Pereira

Committee Member

Vibha Srivastava

Second Committee Member

Fiona Goggin

Third Committee Member

Ainong Shi

Keywords

Biological sciences, Genes, Mutants, Overexpression, Sequencing, Tagging, Transposons

Abstract

Tomato (Solanum lycopersicum) is a crop of immense economic and nutritional importance worldwide and also a good model organism for genomic studies of other dicot species. The recent completion of the tomato genome sequence is a great milestone towards learning about the tomato genome. Elucidation of the function of the different genes using different functional genomic tools is therefore important in adding to this resource. To this end, we have developed an Ac-Ds transposon ‘activation tagging’ (ATag) system to be able to transpose transposon inserts, bearing a strong 35S-enhancer element, all around the genome. An Ac-Ds ATag construct was used to generate transformants in the tomato cultivar M82 that has an erect determinate habit, suitable for greenhouse and field screening. The progeny of putative tomato transformants were germinated and grown to maturity in the greenhouse. Plants with obvious mutant phenotypes were identified, which included dwarfism, altered leaf morphology and necrotic spots on leaves. Presence of the ATag transformed construct was confirmed in the plants by genomic PCR using primers specific to different parts of the Ac/Ds cassette. Activity of the transposon system was also tested by excision PCR using primers flanking the Ds insert in the construct. Insertion sites of the Ds ATag were determined using TAIL-PCR for the progeny plants, and the tagged genes in two mutants were identified by alignment of the flanking sites to the tomato genome. With the availability of the tomato genome sequence, the mutants described will be a good resource for the identification of genes for plant development and tomato breeding.

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