Identification Of Microrna In Peach And Analysis Of The Interactive Mode Between Them And Their Targets

MicroRNA is a class of small endogenous non-coding RNA molecules, length of 19-24nt, widespread in the eukarytic genome. The current study shows that miRNA play an important role at the transcriptional and post-transcriptional levels. miRNA interact with its target mRNAs by a "slicing" or translational inhibitory through sequence complementarity to regulate their expression. miRNAs plays a crucial role in a variety of biological and metabolic pathways in plant, such as leaf morphogenesis and differentiation, identification and differentiation of floral organs, organ development, signaling pathways and biotic and abiotic stress responses et al. Peach originated in China’s western region, is an economically important fruit crops widely distributed in the word. With the rapid development of molecular biology, a large number of peach EST sequences have been released and the entire genomes of peach cultivars’Lovell’ have publically on April 1,2010 (http://www.rosaceae.org/peach/genome). These resources provide a basis for us carry out peach miRNA related research. In order to understand the presence of peach miRNA and its roles in the whole peach growth process, we used peach cv.’Yoshihime’as the experimental material, by bioinformatics and combined with miR-RACE, qRT-PCR, PPM-, RLM-RACE and other experimental techniques, identified the miRNA in peach and further analysis the action mode between miRNA and their target gene.By using bioinformatics methods, twenty-two potential miRNAs from seven miRNA families were first predicted from more than 80,857 EST sequences of peach (Prunus persica). Using two specific 5 and 3 miRNA RACE (miR-RACE) PCR reactions and sequence-directed cloning, we accurately determined the precise sequences, especially both ends, of eight candidate miRNAs. The sequencing results demonstrated that the ppe-miRNAs were conserved to those that were predicted computationally except ppe-miR171b. We validated the existence of two members (ppe-miR171a and miR171b) of the miR171 family in peach that belonged to different precursors. qRT-PCR was further employed in analyzing expression of the eight miRNAs in peach leaves, flowers, and fruits at different developing stages, where some of the miRNAs showed tissue-specific expression.Using bioinformatics approach, we predicted 262 potential microRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5 and 3 miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique peach miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs in peach leaves, flowers and fruits at different stages of development, where the miRNAs showed tissue-specific expression.To further understanding the role of miRNA in peach growing development process, we predicted a large number of peach miRNA’s targets thought bioinformatics methods, we also validated the facticity of this targets and further study the interaction model between miRNA and their targets. In this study we validated the ORF sequence of nine targets by clone and further analysis the interaction pattern of nine targets gene of seven ppe-miRNAs by the methods of PPM-、RLM-RACE and qRT-PCR which development by our laboratory. Research results showed that seven ppe-miRNAs negatively regulate nine targets genes by cleavage pattern. PPM、RLM-RACE results revealed that nine targets gene all have cleavage site between miRNA’s ten or eleven base from the 5 end of ppe-miRNA. qRT-PCR result discovered that the expression of ppe-miRNA and their target genes in different orgen’s have inverse tends. The cleavage production of target gene’s two ends has identical expression tends with their corresponding ppe-miRNAs but has inverse expression tends with the target genes. This result further confirmed the cleavage model between ppe-miRNA and their targets. Research showed that the target genes of miRNAs were invoved in various process of plant growth and development, so validated the facticity of target genes of miRNA and understand the interaction pattern between them have important meaning for we know more about plant life process. This study will help us know more about miRNA’s fuctions in peach.