Isolation by PCR-based methods of a plant antifungal polygalacturonase-inhibiting protein gene

Abstract

A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple fruit was cloned by degenerate oligo-primed polymerase chain reaction (PCR) and Inverse PCR. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design Inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for Inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. A new set of PCR primers were designed to the 5' and 3' ends of the gene, which allowed amplification of the fulllength gene from apple genomic DNA. This method has broad application to isolation of homologues of any gene for which some sequence information is known.