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Molecular analysis of microRNA-target gene interactions in the pine seed
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Pinus pinaster is a conifer species with high ecologic and economic value which covers a vast area of the Mediterranean region. However, the area occupied by P. pinaster has been decreasing mainly due to environmental causes. Somatic embryogenesis may contribute to circumvent this problem as it allows the large-scale propagation of plants adapted to several stresses. However, this technique is poorly understood in P. pinaster and the study of gene expression regulators like miRNAs may contribute to its improvement.
MiRNAs regulate gene expression at post-transcriptional level by mRNA cleavage or translation repression. In this work, a set of four miRNA-mRNA target cleavage interactions were studied. First, mRNAs targets were predicted for a list of miRNAs using bioinformatics tools to analyze available sequencing data, and seven miRNA-mRNA pairs were selected. Second, the expression of four of the mRNAs were analyzed by RT-PCR in two embryo developmental stages, T4B (pre-cotyledonary) and T7 (mature). The four transcripts included ARF 10, 16 ou 17 (regulated by miR160), F-box (regulated by miR482a), LEA (regulated by miRM09664) and PPR (regulated by miRM06658). All transcripts show a higher expression in T4B than in T7 stage. Several parameters were studied to ensure that high-confidence pairs were selected, namely conservation of miRNA and respective target, complementarity between miRNA and respective target, relation between miRNA expression and target expression. Only miRNA160-ARF target fulfilled all the conditions.
It was the first time that potential miRNA targets were identified in P. pinaster. This work contributed to uncover the miRNA landscape in conifers, pointing to potential functions in embryo development. However, more experiments are needed to further validate the interaction between the miRNA-mRNA targets studied.
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Pinus pinaster Embryogenesis regulation miRNA mRNA target PARE RT-qPCR