Computational Identification And Experimental Validation Of MicroRNAs In Eggplant(Solanum Melongena) And Tobacco(Nicotiana Tomentosiformis)

MicroRNAs (miRNAs) are a class of non-protein-coding, endogenous, single-strand molecules of21–24nucleotides in length, which have been reported to regulate gene expression negatively at thepost-transcriptional levels in plants, animals, and other diverse eukaryotes as well as a number of DNAviruses. In plants, miRNAs function to control tissue (leaf, root, stem, and flower) differentiation anddevelopment, signal transduction, and the response to biotic and abiotic stress. In this study, acomputational homology search approach based on the conserved miRNA sequences and the stem-loophairpin secondary structures of miRNAs was adopted in the eggplant expressed sequence tags (EST)databases and the genome of the Nicotiana tomentosiformis. The miRNA and their putative target geneswere analyzed following the diversity of the miRNA families, miRNA clusters, antisense miRNAs andthe functions of the predicted target genes. Several of the predicted Nicotiana tomentosiformis miRNAswere experimentally validated by the RT-PCR method and the target genes of Nicotianatomentosiformis were detected by a modified5′RACE. Using the method described above, we couldlay the foundation for the study of the other plant species especially the solanaceae plants′microRNAs,explore the regulation mechanism of tobacco′s growth and development and provide theoretical basisfor resistance breeding. The main results are as follows:(1)A total of16potential miRNAs belonging to12families were identified in the eggplant ESTdatabase. Three pairs of sense and antisense strand eggplant miRNAs belonging to three differentmiRNA families were also found. Furthermore, miR390and miR399sense/antisense pairs has beenidentified for the first time in plants. Using online software psRNATarget, we further predicted thetarget genes of these16miRNAs and got71potential targets genes on base of15eggplant miRNAs.Most of these target genes were predicted to encode proteins that played key role in eggplant growth,development, metabolism, and stress responses.(2)A total of206high-confidence miRNAs belonged to37families and234putative targetsgenes were identified on base of152miRNAs in genome of the Nicotiana tomentosiformis. EightmiRNA clusters belonged to three miRNA families (miR169,miR156/157and miR1043) and twelvepairs of sense and antisense strand miRNAs belonged to four miRNA families (miR169,miR172,miR396and miR399) were found. InterPro and GO annotations revealed that most of these target genesencoded transcription factors, metabolic enzymes and proteins related to signal transduction or stressresponses. MicroRNAs take part in a wide variety of plant metabolic and biological processes and havean important regulatory function in morphological establishment of plant organs, signal transductionand tolerance to environmental stresses.(3)We verified and profiled the125highly conserved miRNAs in Nicotiana tomentosiformisusing the RT-PCR. The results revealed that the miR319₃was failed to verify and the other miRNAshad different expression patterns. Some species specific, and condition-specific (tissue specific ordevelopmental specific) miRNAs were found. We randomly selected20miRNA: target pairs belongedto14miRNA families and verified them by a modified5′RACE. Our results showed that15miRNA: target pairs belonged to10miRNA families were detected which indicated that predicated target geneswere highly reliable.