PCR Amplification of Persimmon Fruit β-Galactosidase

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We are studying β-galactosidase (EC 3.2.1.23) in softening persimmon fruit (Diospyros kaki L.f. cv Fuyu) and hope to decrease the rate of softening by inserting an antisense construct of the β-galactosidase gene. The N-terminal amino acid sequence of persimmon fruit β-galactosidase was recently reported. Here we report the cloning of a putative β-galactosidase gene from persimmons. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence. 5′-RACE (rapid amplification of cDNA ends) was done using persimmon Poly A+ mRNA extracted using a phenol: chloroform/LiCl method. Purification was done on an oligo dT-cellulose column. A fragment of roughly 150 base pairs was purified by agarose gel electrophoresis and subcloned into the pCR-Script cloning vector from Stratagene. After sequencing and verifying the insert's identity, it will be isolated and used to screen a persimmon fruit cDNA library currently being constructed. Ultimately this cDNA clone will be used to make an antisense β-galactosidase construct that will be transformed into persimmon.