Towards the production of a mutant stylar ribonuclease that inhibits the self-incompatibility response
Document Type Dissertation/Thesis
The mechanism of self-incompatibility (SI) in Solanaceae involves a ribonuclease present in the style of the flower that is capable of recognizing and destroying self pollen, thereby preventing self-fertilization. However, mutations in the enzyme can cause self-compatibility, allowing self-fertilization in some plants. The mechanism of the 51 ribonuclease (S-RNase), and the enzyme's overall role within the SI response, is not well understood, and we seek to gain information about the S-RNase activity through structural and functional comparisons between a mutant and a wild-type enzyme. In order to study the S-RNase, it was decided to synthesize the enzyme in vitro by cloning of the S-RNase gene into an expression host system, avoiding the complications of protein extraction and purification from plant tissue. Before this study, a gene for a mutant S-RNase was synthesized by assembling oligonucleotide fragments. This full gene was successfully ligated into a non-expression plasmid vector, which was then transformed into host Escherichia coli cells. In order to insert the gene iota an expression plasmid, restriction enzyme digestions were carried out to remove the gene but were not successful. DNA sequencing of the gene revealed the restriction enzyme recognition site was incorrect, and site-directed mutagenesis was employed to fix the sequence. Lastly, digestion with other restriction enzymes was conducted, but subsequent ligation into a plasmid vector and transformation into E. coli was also unsuccessful. Cloning of the gene into an expression host is needed for over-expression of the mutant S-RNase. Then, protein extraction and purification can give enough protein to conduct assays comparing mutant and wild type protein structural and functional features to shed light onto the S-RNase mechanism and its role in the SI response.