Computational Identification Of Micrornas In Apple Expressed Sequence Tags And Validation Of Their Precise Sequences By Mir-Race

Plant miRNAs are19to24nucleotide long endogenous non-coding regulatory RNAs that are widely distributed in plant genomes. miRNAs belong to a class of eukaryotic gene expression of negative regulation factors acting at the post transcriptional level, mainly by repressing gene translation or degrading targeted mRNAs. miRNA plays an important role in controlling plant development, flowering timing, metabolism and stress responses. The conservation of microRNA sequences lays a sound foundation for studying the effect of miRNAs on non-model plants whose genomes are unknown. Although there are numerous reports on miRNAs, there is dearth of information on validated apple miRNA.Malus domestica is one of the most economically important fruit crops worldwide. In addition to its economic value, the availability of a large number of expressed sequence tags (ESTs) of M. domestica makes it an excellent source of experimental material for elucidation of gene expression.In this study, apple was used as the experimental material to predict miRNAs and their target genes by bioinformatics. The miRNA and the target gene expression in Malus domestica were systematically and comprehensively studied by qRT-PCR. We used an efficient way to bioinformatically identify mdo-miRNAs by prediction of the precise sequence. The major findings are as follows:1. A comprehensive strategy for identifying new miRNA homologs by mining the repository of available Malus domestica expressed sequence tags (ESTs) was developed. By adopting a range of filtering criteria, we identified a total of31potential Malus domestica miRNAs from more than324,000EST sequences. These mdo-miRNAs belong to16families namely mdo-miR156, mdo-miR162, mdo-miR167, mdo-miR172, mdo-miR393, mdo-miR394, mdo-miR398, mdo-miR399, mdo-miR477, mdo-miR482, mdo-miR828, mdo-miR845, mdo-miR1535, mdo-miR1888, mdo-miR1046and mdo-miR2101. 2. We developed an integrated approach (miR-RACE), combining the strategies of a miRNA-enriched library preparation, two special PCR reactions, and sequence-directed cloning aimed at determining sequences of the miRNAs predicted computationally. This approach was utilized to validate the precise sequences of16Malus domestica miRNAs. Among the16mdo-miRNAs analyzed and validated in this study, only validated mdo-miRNA1535is different from that predicted computationally since their second last nucleotides from the3’ends are different.3. Through the use of the potential miRNA sequences and BLAST searching on the mRNA database of NCBI, we predicted12mdo-miRNA target genes. Functional analysis of these target genes indicate that they play an important role in apple growth and development processes.4. Expression of miRNAs and their target genes during development and maturation of Malus domestica in relation to growth of different tissues was studied where target genes of sixteen mdo-miRNA were predicted and real time PCR used to determine the tissue expression of these sixteen mdo-miRNA and their target genes in the different stages of stems, leaves (young leaves and old leaves), flowers (flower bud and flower) and fruits (diameter1.5cm and4cm). Results show that the expression of mdo-miRNAs and their target genes exhibit an inverse relationship in different developmental stages and different organs.