Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

ISOLATION OF DURUM WHEAT LIPOXYGENASE GENE

MASSARDO D.R.*, DEL GIUDICE A.*, BORRELLI G.M.**, DI FONZO N.**, DEL GIUDICE L.*

* Istituto Internazionale di Genetica e Biofisica–CNR, Via G. Marconi 10, 80125 Napoli

delgiudi@iigbna.iigb.na.cnr.it

** Istituto Sperimentale per la Cerealicoltura, Sezione di Foggia, MIPAF, Foggia

durum wheat, genomic library, lipoxygenase gene, heterologous hybridisation

Lipoxygenases (Loxs) (linoleate: oxygen oxidoreductase, EC 1.13.11.12) are a group of enzymes widely distributed in plants and animals which catalyze the regio- and stereo- selective dioxygenation of polynsaturated fatty acid or their esters containing a cis, cis-1,4-pentadiene system to form monohydroperoxides as primary products. Their occurrence in plants is thought to play a role in fruit ripening and abscission, senescence, and resistance to plant pathogens. Some lipoxygenase isoenzymes may also function as vegetative storage proteins (Siedow 1991, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42, 145-188).

The involvement of Lox in determining the quality of wheat products, particularly with regard to the yellow pigment content, which is decreased during processing of the milled durum product, semolina, into the pasta product, has been widely studied previously (Manna et al. 1998, Cereal Res. Commun., 26, 23-30; Borrelli et al. 1999, Cereal Chem., 76, 335-340). These characteristics makes it an interesting candidate for study.

As an approach to understanding how Lox is regulated, we have undertaken a tentative to isolate Lox gene(s) by screening a durum wheat genomic library with a heterologous Lox sequence gene-probe.

In our present work a Lambda EMBL4/EcoRI durum wheat (cv Trinakria) genomic library, provided by P. Rampino, was screened by plaque hybridisation with the isolated 1200 bp. Almond (Prunus dulcis) Lox gene insert from pAl-Lox1200 recombinant plasmid provided by G. Mita and A. Santino, using standard conditions (Sambrook et al. 1989, CSHL Press).

We have analysed about 2 x 10⁶ plaques probed with the 1200 bp Lox insert. From preliminary screening we have isolated 16 plaque clones which gave a heavy hybridisation signal. Work is in progress for subcloning and DNA sequencing of inserts of these 16 clones. DNA sequences will be searched for homology against the EMBL + Gene Bank Release 78 to verify the percentage homology of the inserts with known plant Lox gene sequences.

Acknowledgement: Work was supported by MIPAF special grant “M.I.C.I.A.”.