Identification And Analysis Of MicroRNA Genes From Solanum Tuberosum

MicroRNAs (miRNAs) are an extensive class of endogenous non-coding RNAs (ncRNAs) found in eukaryotic organisms, which play important roles in many biological processes as posttranscriptional regulators of gene expression. Although several hundred miRNAs have been predicted and identified by computational and experimentally methods both in animals and plants, search for new miRNAs is still a hot academic domain because miRNAs identified so far are far from saturated based on theoretical prediction. Potao (Solarium tuberosum L.) is the fourth biggest crop that can be used as grain and vegetables in the world. However, viruses and diseases are considered worldwide as the main cause of potato severe agricultural loss and seed degeneration. One recent survey has indicated that miRNAs are associated with every stage of plant's growth and development, particularly in response to pathogen invasion and environmental stress. Therefore, predictation and identification of potato miRNAs and their targets have been the subject of considerable interest.We have applied computational RNomics to predict potato miRNAs from expressed sequence tags (EST), genomic survey sequence (GSS), and non-redundant (nr) databases. To validate the prediction of miRNAs in potato, we performed RT-PCR assay to verify expression of mature miRNAs. Finally, we used miRU to predict the potential target genes for these miRNA candidates. The main results are described as below:1. We identified 48 potential miRNAs in potato. Among them, 30 miRNAs were identified in EST database, 11 miRNAs in GSS database and 7 miRNAs in nr database. Our results indicate that the majority of potential miRNAs have homologues in previously known A. thaliana and O. sativa.2. Our results indicate that several miRNAs are conserved among families of dicots, such as miR403, miR869, and miR870. And we found that only these mature miRNAs are highly conserved in plants instead of miRNA precursors.3. Additionally, we have predicted 136 conserved miRNAs in 44 plant species by microHARVESTER which was utilized on line. Most of these potential miRNAs have higher MFEI values(>0.7).4. We found 186 potential targets in potato. Of these predicted targets,～20% are associated with the members of the transcription factor gene families with known or predicted functions, mostly in development, and～30% are related to metabolic and cellular processes, signal transduction, defense/stress functions and their own biogenesis. We also identified a significant number(～24%) of the predicted targets with unknown functions, suggesting possible new roles for conserved miRNAs in potato.5. The efficient and economical method for isolation of plant RNAs was developed based on previous methods. The result of agarose gel electrophoresis of extracted RNA from potato root showed that the improved method was most efficient compared with guanidine isothiocyanate solution and the FastRNA Green method. Successful RT-PCR showed that the RNAs isolated with this method were in good integrity and purity, and can satisfy the needs of most molecular biological experiments.6. Performing RT-PCR assay of 12 miRNAs, we found that several miRNAs have different expression level among different organs, such as miR157a, miR390, miR414a, and miR395. These results suggest that the examined miRNAs have diverse expression patterns during development.