Identification and validation of potential conserved microRNAs and their targets in peach (Prunus persica)

Abstract

MicroRNAs are a class of small, endogenous, non-coding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many plant species, especially from model plants and crops, they remain largely unknown in peach. In this study, 110 potential miRNAs belonging to 37 families were identified using computational methods. A total of 43 potential targets were found for 21 families based on near-perfect or perfect complementarity between the plant miRNA and the target sequences. A majority of the targets were transcription factors which play important roles in peach development. qRT-PCR analysis of RNA samples prepared from different peach tissues for 25 miRNA families revealed that miRNAs were differentially expressed in different tissues. Furthermore, two target genes were experimentally verified by detection of the miRNA-mediated mRNA cleavage sites in peach using RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE). Finally, we studied the expression pattern of the two target genes in three different tissues of peach to further understand the mechanism of the interaction between miRNAs and their target genes.

Fig. 1

Analysis of potential miRNAs in peach (A) miRNA family in peach (B) Size distribution of miRNAs in peach (C) Number of peach miRNAs against different lengths of mature miRNAs.

Fig. 2

Stem-loop hairpin structures of representative miRNA families. Red indicates mature miRNA sequences.

Fig. 3

Relative expression levels of peach miRNAs in different tissues.

Fig. 4

Mapping of mRNA cleavage sites by RNA ligase-mediated 5′ RACE. Each top strand depicts a miRNA-complementary site in the target mRNA, and each bottom strand depicts the miRNA. Watson-Crick pairing (vertical dashes), G:U wobble pairing (circles) and mismatched base pairing (X) are indicated. Arrows indicate the 5′ termini of mRNA fragments isolated from peach. Numbers indicate the fraction of terminating cloned PCR products.

Fig. 5

Relative expression levels of peach miRNA target genes in different tissues.