We focus on three genes involved in accumulating sugars in different phases of tomato fruit growth and ripening: LIN5, AGPL1 and TIV1 by studying their transcriptional regulation, in particular by the Cis-regulatory elements (CREs) in their promoters. CREs define the spatial and/or temporal expression of their gene. It has been proposed that by targeting this part of genes’ transcriptional regulation new phenotypes can be developed without harmful pleiotropic effects, and indeed much natural variation selected for in crops is variation in these rather than in protein coding regions. A major hurdle in this endeavor is the lack of detailed knowledge on how CREs work. We will perform a comprehensive study on CREs of the sugar-related genes during tomato fruit development by using CRISPR/Cas9 mutagenesis to create variation through systematic deletion of promoter regions. By studying the effect of mutations on the target gene expression, we intend to discover new types of CRE functionalities in an in vivo system. Combining the mutagenesis method with proven methods for studying CRE function, we will work towards a comprehensive model explaining the transcriptional regulation of the three target genes. This knowledge will help us to work towards a better tasting tomato.
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- CRISPR mutagenesis in tomato (Cloning constructs, tomato transformation, tissue culture)
- Genetic analysis of tomato mutants
- Gene expression analysis (RNA isolation, microarrays, qRT-PCR)
- Phenotyping (Brix analysis, metabolomics)
- Protein-DNA and protein-protein interactions (ChIP-seq, yeast 1 and 2-hybrid analysis)
- Good theoretical and practical basis in (plant) molecular biology