Cloning of a cDNA encoding phospholipase D from Pimpinella brachycarpa

Phospholipase D (PLD, EC 3.1.4.4) has been known to be related to various cellular processes in plants. To gain an understanding of the property of the enzyme in Pimpinella brachycarpa, the cDNA of the enzyme was isolated by PCR with degenerate primers, cDNA library screening, and 5' RACE. The full-length PLD cDNA is 2859 bp long and contains an open reading frame of 2424 bp coding for a polypeptide of 808 amino acids. The deduced enzyme has a calculated molecular mass of 91.7 kDa and pI of 5.86. The percent identity and similarity values of P. brachycarpa PLD with those of other PLDs in plants are 70 approximately 78 and 84 approximately 95, respectively. It was identified that PLD from P. brachycarpa has HQKIVVVD and HAKMMIVD sequences which were homologous with a duplicated HXKXXXXD motif that has been conserved in PLDs from plants, animals, and yeast. Based on the analysis of amino acid similarity, it is believed that PLD from P. brachycarpa is an alpha form which is distinct from PLD beta reported recently. The N-terminus is homologous to the C2 domain which is present in a number of different proteins involved in signal transduction and membrane trafficking in animals. Southern and northern blot analyses indicated that PLD was expressed from one copy of PLD gene in the genome of P. brachycarpa.