PCR AMPLIFICATION AND CLONING OF AVOCADO BETA-GALACTOSIDASE
SUE HUM-MUSSER, David A. Starrett, Nathan M. Springer & Kenneth C. Gross. Biol. Dept., Southeast Missouri State Univ., Cape Girardeau, MO 63701 (SHM, DAS, NMS). USDA, ARS, Hort. Crops Quality Lab., Beltsville, MD 20705 (KCG).
Roughly half of the avocado fruit harvested do not reach the consumer due to premature ripening and related spoilage. In order to gain a better understanding of avocado ripening we are investigating the softening process. We are interested in beta-galactosidase from avocado mesocarp which is able to solubilize tomato fruit pectin (De Veau, et al., 1993, Physiologia Plantarum) and which is detected early in ripening before the appearance of cellulase and polygalacturonase. Beta-Galactosidase activity also increases in various fruits during ripening. We intend to screen a cDNA library of ripening avocado (Persea americana Mill. cv. Hass) fruit to isolate and sequence a full-length cDNA clone encoding beta-galactosidase. Previously, a fragment of beta-galactosidase was PCR amplified using degenerate oligonucleotide primers based on heterologous plant beta-galactosidase sequences. The fragment was subcloned into the pCR-Script cloning vector (Stratagene, La Jolla, CA), characterized and sequenced. The insert will be purified, identity verified, and non-isotopically labeled using the Genius System (Boehringer- Mannheim, Indianapolis, IN). We will then screen a previously constructed cDNA library from ripening avocado fruit contained in Zap II (Stratagene). The full length clone of avocado beta-galactosidase is expected to be between 2 and 2.5 kb in length based on the lengths of other plant beta-galactosidases. Once the full-length cDNA clone is isolated and purified, it will characterized and commercially sequenced. Potential applications of this work include construction of an antisense clone that will be transformed into fruit which utilize beta-galactosidase as an important enzyme in the ripening process. This may potentially delay or inhibit softening, resulting in an increased shelf life by reducing losses due to premature ripening.