Plant Biotechnology
Molecular Biology and Genetics
EJB Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.2 No.3, Issue of December 15, 1999.
© 1999 by Universidad Católica de Valparaíso -- Chile Received October 8, 1999 / Accepted October 21, 1999
RESEARCH ARTICLE

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

Melanie S. Arendse
Biotechnology Division, Agricultural Research Council-Roodeplaat
Vegetable and Ornamental Plant Institute,
Private Bag X293, Pretoria, 0001, South Africa.
E-mail : melanie@vopi.agric.za

Ian A. Dubery
Department of Biochemistry
Rand Afrikaans University,
P.O. Box 524, Auckland Park, 2006, South Africa.
E-mail : iad@na.rau.ac.za

David K. Berger*
Biotechnology Division, Agricultural Research Council-Roodeplaat
Vegetable and Ornamental Plant Institute,
Private Bag X293, Pretoria, 0001, South Africa.
E-mail : dberger@vopi.agric.za

* Corresponding author

Keywords : Degenerate, Inverse polymerase chain reaction, Polygalacturonase-inhibiting protein.


Abstract Full Text

A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple plants 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 full-length gene from apple genomic DNA. This method has broad application to isolation of homologues of any gene for which some sequence information is known.

Supported by UNESCO / MIRCEN network
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