PCR AMPLIFICATION AND CLONING OF AVOCADO ALPHA-GALACTOSIDASE
1NATHAN M. SPRINGER, 1David A. Starrett, 1Sue Hum-Musser & 2Kenneth C. Gross. 1Biol. Dept., Southeast Missouri State. Univ., Cape Girardeau, MO 63701. 2USDA, ARS, Horticultural Crops Quality Lab., Beltsville, MD 20705.
Large amounts of avocado fruit are lost every year due to premature ripening and related spoilage. Pectin solubilization and its effect on softening is an important aspect of the ripening process. Avocado alpha-galactosidase has been shown to solubilize pectin, a major component of fruit cell walls (De Veau, et al., 1993, Physiologia Plantarum). We are attempting to clone avocado alpha-galactosidase in an effort to determine its involvement in the ripening process. Heterologous degenerate oligonucleotide primers were constructed by using conserved regions of coffee (Zhu and Goldstein, 1994, Gene) and guar (Overbeeke et al, 1989, Plant Mol. Biol.) alpha-galactosidase genes. These primers were then used to PCR amplify avocado (Persea americana Mill. cv. Hass) cDNA. The resulting fragments were cloned into the pCR-Script plasmid vector (Stratagene, La Jolla, CA), characterized, and sequenced. The sequence revealed a high degree of homology with the coffee and guar alpha-galactosidase sequences. The inserts were then excised, purified, and labeled using a Genius System non-isotopic labeling kit (Boehringer-Mannhiem, Indianapolis, IN). The labeled probes are being used to screen a cDNA avocado library constructed from ripening fruit. Positive plaques will be isolated, the insert subcloned, characterized and the sequence determined. This work will eventually allow for the construction of antisense fragments for alpha-galactosidase. These antisense constructs used alone and in conjunction with other antisense constructs will help to elucidate potential roles of alpha-galactosidase in ripening related events in avocado.