Cloning of the S₈-RNase (S₈-allele) of Japanese Pear (Pyrus pyrifolia Nakai)

Abstract

The expression of S₈-RNase was confirmed in pistils of two Japanese pear cultivars, ‘Ichiharawase’ (S₁S₈) and ‘Heiwa’ (S₄S₈). The complete sequence of the S₈-RNase gene was determined connecting the nucleotide sequences of partial cDNA and 5' terminal genomic DNA fragments amplified by RT-PCR and genomic PCR. The S₈-RNase has an open reading frame of 684 nucleotides encoding 228 amino acid residues. A hypervariable region (HV) of S₈-RNase, which is quite different from those of S₁- to S₇-RNases, includes an intron of 234 bp. The similarity of deduced amino acid sequences between S₈-RNase and the seven S-RNases of Japanese pear ranged from 56.7% (S₃-RNase) to 70.2% (S₇-RNase). Based on its nucleotide sequence, we selected NruI as S₈-RNase specific restriction endonuclease and established the PCR-RFLP system for discriminating S₁- to S₈-alleles.