Abstract:

PCR were applied for point mutagenesis of MxIRT1 gene. Two pairs of primers were designed according to the Opening Read Frame (ORF) and sequence for mutation of MxIRT1. Two fragments overlapping at the target mutated base were amplified by PCR. The fragments were amplified after dilution with the primers designed with the two ends of ORF, and the two mutated fragments were stringed together. The annealing temperature and extension time were optimized for successful amplification. The mutated MxIRT1 were ligated into pEASY-T2. Sequencing result showed that we mutated MxIRT1 successfully. DNA recollection and purification were not necessary for this experiment. We establish an efficient, simple gene mutagenesis system.

Key words: Malus xiaojinensis, MxIRT1, PCR, Site Direct Mutagenesis