GENETICS AND EXPRESSION OF 2 PECTINESTERASE GENES IN VALENCIA ORANGE

The genetics and expression of pectinesterase (PE) genes were examined in Valencia orange. Degenerate primers based on partial amino acid se quence of a 36 kDa PE protein isolated from juice vesicles were used t o amplify a 350 bp DNA fragment from cDNA prepared from juice vesicle total RNA. Two groups of 350 bp PE clones with 66% sequence identity w ere isolated. A clone from each group was used to screen a Valencia or ange genomic DNA lambda library. Two different lambda clones that cont ained complete PE coding sequence (CsPME1 and CsPME3) and a third lamb da clone that contained partial PE sequence (CsPME2) were characterize d. The CsPME1 gene contained two exons (1063 and 689 bp) interrupted b y a 1452 bp intron, whereas the CsPME3 gene had two exons (844 and 686 bp) interrupted by a 771 bp intron, CsPME1 shared significant sequenc e similarity with the partial clone CsPME2, including the entire clone d region of the first exon, a large region in the 5' portion of thr in tron and the 3' portion of tile second exon, but the 3' portion of the intron and the 5' portion of the second exon were dissimilar. Souther n blots suggested that Valencia orange has two genes within each PE gr oup. Full-length cDNA clones that shared 99% sequence identity with Cs PME1 AND CsPME3 were isolated. Both groups of PE genes were differenti ally expressed in tissues of Valencia orange, and in addition CsPME3 a ppeared to be ethylene-regulated. The deduced proteins of PE cDNA clon es CsPME1 (63.5 kDa) and CsPME3 (56.3 kDa) were considerably larger th an the PE protein we isolated from Valencia orange juice vesicles and also other mature plant PE proteins. The estimated size of group I (2. 2 kb) and group II (2.0 kb) PE mRNAs also predicted a larger protein t han was isolated from juice vesicles. Alignment of the mature tomato a nd mung bean PE proteins, the most N-terminal sequence we obtained fro m polypeptides derived from the 36 kDa PE isolated from juice vesicles and the deduced amino acid sequences of plant PE cDNA clones suggest that a post-translational cleavage event separates the variable N-term inus from the more conserved C-terminal domain of the mature PE protei n.