Enjoying the fruits of knowledge: cracking the CIS-regulatory code of gene regulation
What makes a nice tomato? An important aspect of tomato quality generally liked by consumers is sweetness, created by sugars. Creating a tomato with a higher sugar content is the focus of my PhD project.
I focus on two genes involved in accumulating sugars in different phases of tomato fruit growth and ripening: LIN5 and AGPL1 by studying their transcriptional regulation, in particular by the Cis-regulatory elements (CREs) in their promoters. CREs, small motifs in the DNA, define the spatial and/or temporal expression of genes. 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 CREs rather than in protein coding regions. A major hurdle in this endeavour is the lack of detailed knowledge on how CREs work.
In my PhD project, we 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 and sweetness phenotype, 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.
Are you interested in this work? Then you are welcome to do a BSc or MSc thesis! Depending on your interests and ideas, we can discuss several specific projects, ranging from 18-39 ECTS. Are you a molecular hero and you want to explore the possibilities of working with CRISPR? Then you can try to upgrade the tomato with homology-directed gene targeting. Do you love working with plants? A bunch of interesting CRISPR/Cas9 mutant tomato plants are calling for in depth analysis! You can contact me through the contact form.
CRISPR mutagenesis in tomato (Cloning constructs, tomato transformation, tissue culture)
Genetic analysis of tomato mutants
Gene expression analysis (RNA isolation, qPCR)
Phenotyping (Brix analysis, metabolomics)
Protein-DNA and protein-protein interactions (yeast-1-hybrid analysis, Dual Luciferase Assays)
Good theoretical and practical basis in (plant) molecular biology