MicroRNAs and target genes involved in E. globulus xylogenesis: in silico prediction and experimental validation

Abstract

Portugal is one of the largest producers of pulp and paper derived from Eucalyptus globulus, which makes this a valuable species for the country. Wood is a complex and variable material, and its posttranscriptional regulation knowledge is only beginning. MicroRNAs (miRNA) are small size (21-24nt), endogenous non-coding RNAs, involved in post-transcriptional regulation. MiRBase v20 database encloses thousands of entries, however none from Eucalyptus. In this study we aim to validate E. globulus miRNAs candidates; to predict in silico and validate experimentally the miRNAs targets; and analyze the gene expression of validated targets. Four miRCa-02, miRCa-04, miRCa-08 and miRCa-09 candidates were validated by Northern blot and there in silico prediction revealed 42 target genes. Fourteen predicted target genes were tested through the RLM 5’-RACE methodology, but only three predicted targets were validated (Eucgr.E01509, Eucgr.C01382 and Eucgr.J02113 predicted target genes for miR171, miRCa-04 and miRCa-08, respectively). Expression of these three target genes analyzed by RT-qPCR suggests that the distinct expression levels found may be related with to wood formation in Eucalyptus globulus. For the first time, four Eucalytus miRNAs and their target genes were disclosed and validated by bioinformatic and molecular tools.