Characterization of target mRNAs for Prunus persica microRNAs using an integrated strategy of PLM-RACE, PPM-RACE and qRT-PCR

Abstract

MicroRNAs (miRNAs) in higher plants control their target mRNAs by either mRNA cleavage or translation repression at transcriptional or posttranscriptional levels. To verify more predicted target genes of Prunus persica microRNAs (Ppe-miRNAs) and elucidate the modes by which these Ppe-miRNAs work on their target genes, nine potential target genes (ppa005013m, ppa006611m, ppa007056m, ppa007653m, ppa001386m, ppa001781m, ppa003783m, ppa003465m, ppa004699m) for seven Ppe-miRNAs (Ppe-miR156a, Ppe-miR164a, Ppe-miR165a, Ppe-miR171a, Ppe-miR172a, Ppe-miR393a, Ppe-miR394a) were experimentally verified by a new strategy integrating modified 5′ RLM-RACE (RNA ligase-mediated 5′ rapid amplification of cDNA ends), 3′ PPM-RACE (Poly(A) polymerase-mediated 3′ rapid amplification of cDNA ends) and qRT-PCRs of cleavage products. The results show that these Ppe-miRNAs negatively regulate the expression of their target mRNAs by guiding cleavage of corresponding target mRNA with the cleavage sites mostly being at the tenth nucleotide of 5′ end binding sites of Ppe-miRNAs. The expression patterns of Ppe-miRNAs, their target mRNAs and the cleavage fragments of target mRNAs were also analyzed in eight different peach tissues. There was an inverse relationship between expression of Ppe-miRNAs and their target mRNAs in eight tissues, and expression of both cleaved targets and Ppe-miRNAs verify the cleavage mode of Ppe-miRNAs on their target genes. Our results confirm the importance of Ppe-miRNAs in peach growth and development, and further increase understanding on the important regulatory roles of Ppe-miRNAs in peach, this suggest more study on Ppe-miRNAs and targets can enrich the knowledge of miRNA's functions in peach.