Molecular cloning of beta-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening

We have cloned a cDNA fragment encoding a beta -galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-CAL), It contained an untranslated sequen ce of 182 nucleotides at the 5 ' end, a presumptive coding sequence of 2,19 3 nucleotides and an untranslated sequence of 268 nucleotides including a p olyadenylation signal and a poly (A) tail at the 3 ' end, It encoded a prot ein with a calculated molecular weight of 80.9 kDa which consists of 731 am ino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta -galactosida se cDNA, The peptide sequence obtained from the purified Japanese pear beta -galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIIN GQKRILISG (amino acid 25-45), Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA, The mRNA was detecte d solely in the fruit; none was detected in the buds, leaves, roots or shoo ts of the Japanese pear. The steady-state level of the beta -galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regar dless of the cultivar, no JP-CAL mRNA was detected in the immature fruit. I ncrement of the mRNA level with fruit ripening coincided with the increase in the beta -galactosidase III activity. Our results showed that the expres sion of JP-CAL correlated with fruit softening and JP-GAL may be beta -gala ctosidase III.