Design of a Shuttle Vector Using Homologous Recombination for Plant Transformation

A four-system shuttle vector was created for further research into sugar partitioning in the plant phloem system. The shuttle vector of interest was synthesized through homologous recombination in yeast and contains functional components for Saccharomyces cerevisiae, Escherichia coli, and Agrobacterium tumefaciens, and plants. Ultimately, the shuttle vector will carry multiple genes to a target plant and allow for the study of previously unexamined metabolic pathways and physiological functions. The homologous recombination utilized during the experiment was a DNA double-stranded break repair mechanism present in S. cerevisiae that is able to combine multiple plasmid fragments into one complete construct. Our plasmid provides flexibility of gene manipulation that currently cannot be found in plant transformation technology.

This new plasmid construct contains a 3-4.2kb fragment from pRS314, 314, 315, 424, or 425 which contains either the tryptophan, leucine, or histidine amino acid synthesizing gene sequence and the yeast functional components. Furthermore, the construct holds a 1.2kb fragment from pCR8-pNOS-BAR-pA35S, containing a Basta resistance gene and the plant functional components. This vector uses pCAMBIA0390 as its backbone because of its role as a binary vector. These oligonucleotides overlap 40 or 50 base pairs between the right and left side of each DNA fragment. The main advantages of this method over its alternatives are its flexibility and ability of homologous recombination.

To create this vector, a unique plasmid synthesis protocol was implemented. A program, SnapGene, was utilized to provide virtual plasmid maps and cutting sites for restriction enzymes. The original plasmids were digested using the enzymes ScaI-HF, PvuII-HF, BamHI-HF, and EcoI-HF to create fragments containing the functional units for each organism. Following this procedure, a yeast transformation was completed to combine these fragments via homologous recombination during the yeast growth cycle and grown on specific selection medium. The surviving yeast cell DNA was then harvested using the Grab n’ Bust method and electroporated into E. Coli to increase the number of plasmid copies. Once minipreps were created, another digest was done to confirm the construction of the plasmid.

Results gathered from experimentation suggest the presence of the desired shuttle vector and the success of the chosen method. Confirmation of the desired plasmid structure was completed by comparison via image analysis of the SnapGene simulated gel electrophoresis restriction digest and the actual restriction digest. Further research includes sequencing of the DNA samples and increasing efficacy of plasmid synthesis. Future work will focus on the use of this binary vector to deliver genes for the purpose of plant metabolic engineering experiments.