Identification, characterization, and verification of miR399 target gene in grape

The microRNA miR399 plays an important role in phosphorus signal transduction pathways in plants. Previously, miR399 was shown to be closely associated with berry ripening in grape (Vitis vinifera). The objective of the present study was to elucidate the evolutionary charac-teristics of the miR399 gene family in grape and to verify the cleavage effect on the target genes. Grape miR399s were identified by miRNA sequencing and retrieval from the miRBase database. The mature sequences and precursor sequences were subjected to phylogenetic analysis to reconstruct evolutionary trees, as well as secondary structure analysis of the precursor sequence, and prediction of target genes. The cis-acting elements in the miR399 promoter were predicted and the cleavage effect of grape miR399b on its target genes was verified. The grape miR399 family comprised nine precursor sequences and nine mature sequences. The precursor sequences formed a typical and stable stem-loop structure. The minimum folding free energy ranged from -55.70 kcal center dot mol-1 to -37.40 kcal center dot mol-1. Multiple sequence alignment revealed that the miR399 family was highly conserved. The grape miR399 family was phylogenetically closely related to peach, apple, and citrus miR399s. Grape miR399s were predicted to target inorganic phosphate transporter 1-3, phospholipase D delta-like, and beta-glucuronosyltransferase. The cleavage effect of grape miR399b on the target genes was verified by means of a dual-luciferase assay and 50 RLM-RACE. Histochemical GUS staining showed that the promoter activity of miR399b was promoted by GA3 treatment.