Cloning and characterization of a wheat neutral ceramidase gene Ta-CDase.

Ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine and sphingosine-1-phosphate. This study first reports on the molecular cloning, sequencing and expression profile of the gene encoding the wheat neutral ceramidase designated as Ta-CDase. A full length wheat Ta-CDase gene is obtained by rapid amplification of cDNA ends (RACE) based on the sequence of the WSRC36 fragment from an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis f. sp. tritici. The open reading frame (ORF) of 2,839 nucleotides encodes a polypeptide of 785 amino acids with a predicted isoelectric point (pI) of 6.398. The protein conserved domain search indicates that the polypeptide contains the signature of ceramidase, signal peptide sequence and transmembrane region. A phylogenetic analysis reveals that a high degree of relatedness exists among wheat Ta-CDase and ceramidases from other plant species at the amino acid level, while its relationship to that of animals and pathogens is more distant. The expression profile of the Ta-CDase shows a very strong expression of transcripts only at 48 h post inoculation (hpi), while expression level is low at other time points. Southern blot analyses showed that Ta-CDase is a multi-copy gene and located on wheat chromosome 4D and 5A.