Mutagenesis of genes for starch debranching enzyme isoforms in pea by means of zinc-finger endonucleases

Starch debranching enzymes in plants are divided into two groups based on their ability to hydrolyse different substrates. The first group, pullulanases, hydrolyses α-1,6-glucosidic linkages in substrates such as pullulan, amylopectin and glycogen. The second group of debranching enzymes, isoamylases, hydrolyse glycogen and amylopectin and are not active on pullulan. Three isoforms of isoamylase and a pullulanase have been isolated from cDNA library of Pisum sativum. These isoamylases have been characterised based on the their heterologous expression in E coli. Based on the DNA sequence that encodes these debranching enzyme, a specific mutagenesis targeting at these DNA will be attempted. The method that will be employed are based on the techniques developed by Wright et al. (2005). This technique involves the homologous recombination of DNA that is mediated by zinc-finger endonucleases. Vectors will be constructed to include a fragment that will modify these genes. Microinjection technique will be used to insert these vectors into pollen which then will be fertilized. Using this technique, it is hoped that null mutant for each enzyme will be created and the exact role of these enzymes for the synthesis and degradation of starch in plants will be elucidate. (author)