Molecular cloning and characterization of a cDNA encoding a ryegrass (Lolium perenne) ENOD40 homologue

The ENOD40 gene, found in several leguminous and non-leguminous plant species is expressed in the pericycle of legume roots early in the nodulation process, adjacent to the protoxylem poles, but before the cortical cells divide to form the nodule itself. The ENOD40 transcript contains only short open reading frames which give rise to short peptides with a signaling function. The current work reports the cloning and analysis of ENOD40 genes from perennial ryegrass (Lolium perenne) and barley (Hordeum vulgare). Specific polymerase chain reaction (PCR) techniques lead to the isolation of a 659 bp cDNA encoding an ENOD40 homologue, designated LpE-NOD40, from a Lolium perenne stem cDNA library. In addition, a partial ENOD40 cDNA of 384 bp was isolated from barley (Hordeum vulgare) by RT-PCR cloning. The LpENOD40 transcript encodes a putative dodecapeptide, similar to that identified in ENOD40s from leguminous plants and other dicots, and also to ENOD40s from monocots. The coding sequences of ryegrass and barley ENOD40 are represented by a short open reading frame of 12 amino acids. These show a high degree of similarity to each other and to other ENOD40 sequences from monocots. The corresponding genomic DNA from a genomic ryegrass lambda library revealed that the LpENOD40 gene contains no introns. Southern blot analysis shows that the ryegrass genome contains a single copy, possibly two copies, of the gene. Alignment of the ENOD40 cDNA sequences from ryegrass and barley revealed high (77%) nucleotide homology. The ENOD40 peptides are highly conserved, not only among monocots but also on comparison with the dicot peptides. The amino acid identity of region I from ryegrass with its counterparts in maize, barley and rice is 92, 83 and 75% respectively. Expression analysis by RT-PCR demonstrates that a high level of LpENOD40 gene transcript was found expressed in stem tissue, while a lower level was detected in leaves and only a very low expression in flowers of perennial ryegrass. No LpENOD40 transcript was detected in roots.