Purification and Characterization of Phospholipase D (PLD) from Rice (Oryza sativaL.) and Cloning of cDNA for PLD from Rice and Maize (Zea maysL.)

摘要

Phospholipase D (PLD) was purified to high homogeneity from rice bran (Oryza sativaL.). Two peaks of PLD activity were resolved by Mono Q anion-exchange chromatography. The molecular mass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDa in gel filtration. Antibodies raised against the protein in one of the peaks precipitated the enzyme activities in both peaks. Enzymatic characteristics of PLD in the two peaks were identical except for a difference of 0.1 in the isoelectric points. Sequence analysis covering more than 10%of the amino acids of the proteins and peptide mapping did not detect any difference in the primary structure of the proteins. A cDNA for PLD was isolated from rice and it encoded a protein of 812 residues. The N-terminal sequences of purified PLDs matched the deduced amino acid sequence starting from residue 47. A Northern blot showed this gene was expressed in leaves, roots, developing seeds and cultured cells, and a Southern blot detected a single band of rice genomic DNA hybridizing to the cDNA. A cDNA for PLD was also isolated from maize. The similarity of the deduced amino acid sequences of PLD was 90%between rice and maize, 73%between the cereals and castor bean.