Differential expression of tandemly duplicated Enod8 genes in Medicago

Enod8 is a Medicago nodule specific gene that belongs to the GDSL family of lipase and esterase genes found exclusively in plants and bacteria. Genomic blot analysis of Enod8 from both Medicago saliva, alfalfa, and Medicago truncatula, the model legume, demonstrate that Enod8 is present in multiple copies in both plants. Enod8 genes were isolated from two M. truncatula genomic libraries, a lambda based library and a BAC library. Survey sequencing of the BAC clones together with complete sequencing of 12.7 kbp, that includes a highly transcriptionally active Enod8 gene, revealed a cluster of three Enod8 genes. Sequence data also show the existence of at least three and as many as five additional Enod8 genes in close proximity to the cluster. Expression analysis of the cluster of Enod8 genes was done by RNA blot analysis and gene-specific RT-PCR. It showed that two of the genes are transcriptionally active, one much more highly than the second. The third Enod8 gene was not found to be expressed under any conditions tested, is lacking a 5' exon, and is likely to be a pseudogene. Antisera were raised to an Enod8-specific oligopeptide and demonstrated to be Enod8 protein specific. The Enod8 protein expression profile in M. truncatula is consistent with one very active Enod8 gene that is post-translationally modified by N-glycosylation. In contrast, five Enod8 proteins are present in alfalfa nodules, consistent with a much greater Enod8 gene complexity in alfalfa than in M. truncatula. No Enod8 proteins were found in roots. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.