Molecular cloning and characterization of phospholipase D from Jatropha curcas

Phospholipase D (PLD, EC 3.1.4.4) is a key enzyme involved in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress, which was cloned from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 2,886 bp in length with a complete open reading frame of 2,427 bp which encoded a polypeptide of 808 amino acids including a putative signal peptide of 53 amino acid residues and a mature protein of 755 amino acids with a predicted molecular mass of 86 kD and a pI of 5.44, having two highly conserved HKD' motifs. Phylogenetic analysis indicated the J. curcas PLD alpha (JcPLDalpha) showed a high similarity to other PLD alpha from plants. Semi-quantitative RT-PCR analysis revealed that it was especially abundant in root, stem, leaf, endosperm and flower, weakly in seed. And the JcPLDalpha was increasedly expressed in leaf undergoing environmental stress such as salt (300 mM NaCl), drought (30% PEG), cold (4degreeC) and heat (50degreeC). The JcPLDalpha protein was successfully expressed in Escherichia coli and showed high enzymatic activities. Maximal activity was at pH 8 and 60degreeC.