Computational Predication And Experimental Verification Of Novel MiRNA In Tomato

MicroRNAs (miRNAs), ranging in size from 20 to 25 nucleotides, are a class of endogenous regulatory noncoding RNAs that have been found in various eukaryotes. Increasing evidences has shown that miRNAs participate in a variety of regulatory pathways, including development, virus prevention, hematopoiesis, organogenesis, cell proliferation and apoptosis, fat metabolism, etc. Mature miRNAs are processed from longer primary transcripts by a series of ribonuclease, and then incorporated into RNA-induced silencing complex (RISC), in which they recognize target mRNA by base-pairing and guide RISC to cleave the mRNA or inhibit the translation of the mRNA, depending on the complementarity between them.Although many miRNAs have been identified in recent years, only a very small amount of miRNAs of plant species has been discovered and functionally identified. Moreover, plant miRNAs studies have been confined mainly to Arabidopsis and rice, two plant model species with fully sequenced genomes. Tomato(Solanum lycopersicum) is one of the most important economical crops in the world. The research of tomato miRNA is quite few. In our research, according to the conservative characteritic of miRNAs, we want to identify novel miRNAs in tomato through computional prediction and northern blot verification.Firstly, to search potential miRNAs, a total of previously known 1300 miRNAs and their precursor sequences from A. thalianan, et )were obtained from miRNA Registry Database. To avoid the redundant or overlapping miRNAs, the repeated sequences of miRNAs within the above species were removed and the remaining sequences were used as query sequences for BLASTN search. Then we obtain the pre-miRNA from EST and GSS database with BLASTN search. After removal of repeat and protein-coding sequences with BLASTX search, we predict the miRNAs in tomato. A total of 12 potential miRNAs were detected following a range of strict filtering criteria. Among them, 6 miRNAs were identified in EST database and 6 miRNAs in GSS database. Using these potential miRNA sequences, we could further blast the mRNA database and predict the target genes. Finally we found 29 potential targets in tomato.The prediction of potential targets of new miRNAs in tomato shown that there were 10 target ESTs encoding functional proteins, and other 19 target ESTs coding unknown proteins. In order to validate the predicted miRNAs, we extract total RNAs from 6-week old tomato leaves and stems, then enrich small-sized RNAs according to a protocol described in the main text. Then we synthesis the probe according to the 12 miRNAs candidates. The expression patterns of 12 candidates were examined in leaves and stems of tomato seedlings using PAGE/northern blotting method. There are 5 novel miRNA genes confirmed.. These 5 confirmed miRNAs were expressed in both leaves and stems. The other 7 miRNAs have no signal in our test. In this research, we only extract total RNAs from 6-week old tomato leaves and stems. So the other miRNAs candidates may exit in the special growth period and environment stress. Also the missed miRNAs might be present in root, fruit or other organs. More research need to be done in validating the other predicted miRNAs.