Cloning And Characterization Of Micrornas From Barley (Hordeum Vulgare L.)

Barley(Hordeum vulgare L.)is one of the principal cereal crops in the world cultivatedin all temperate areas ranking fifth in world crop production. It's used for animal feed,brewing malts, and human consumption. It has been considered as a model species for geneticanalysis thanks to its widely available genetic information. Therefore, barley can be thereference of wheat genetics and breeding and the source of specific useful genesMcroRNAs(miRNAs)are small class of endogenous RNAs that regulates the geneexpression which is involved in various biological and metabolic processes and stressresisitance at the post-transcriptional level in both plants and animals. Many major crops havebeen traced for miRNAs in past few years, yet miRNAs of barley(Hordeum vulgare L.)arestill questioned.In this study, we identified small RNA transcriptome of barley to uncover novel andspecial microRNAs, sequenced using Solexa sequencing method. Millions reads wereobtained from short RNA libraries generated from pooled total RNA extracted from barley(cv.Clipper)roots, stems, leaves and spikes, after bioinformatics analysis, We identified theconserved and barley-specific miRNA. The main results are as follows.First,10,367,915high quality reads(9,540,562clean reads)are obtained by Solexasequencing method, including4,045,224unique sRNA which the length more than18Nucleotide acid(nt). After blast with genome sequence, in small RNA dataset there are83,300unique Ribosomal RNA accounted1,006,189reads, nd23,642unique repeataccounted90,970reads and3,020small nuclear RNA accounted15,180reads,1,872smallNucleolus RNA accounted6,711reads and16,957transfer ribonucleic acid accounted1,154,444reads. In addition,3,916,433unique unannatation sRNAs have7,267,068reads,accounted76.17percent of total sRNA.Secondly, the unannatation small RNA sequences were used to do a Blastn searchagainst the miRNA databas(emiRBase18.0)in order to identify conserved miRNAs in barley.4742sequence were successful matched the previous211miRNAs, belonging to88families,as a total of543,016reads, of which126miRNA belonging to58families is first founded in barley by us.The third, the genome sequence and all non coding EST were analysis by softwareMireap developed by BGI, corresponding to Criteria of secondary structure, Dicer enzymesites and the minimum free energy, mismatch number and other parameters.133miRNAwere identified that can be divided into50families. All133novel miRNAs are considered tobe species-specific because no homologs were found in other species. In addition,15miRNA*is the first reported by us.The fourth, part of miRNA were validated by stem-loop qRT-PCR. The qRT-PCRresults demonstrate that all tested miRNAs are expressed in barey. However, the expressionlevels of the different miRNAs varied, the average expression level of conserved miRNA isΔCT-0.2, which is much higher than the novel miRNA(ΔCT4.8). This result is the sameas the results of our high throughput analysis.At last, to deepen the knowledge about the functions of the newly identifiedspecies-specific as well as conserved barley miRNAs, putative targets of these miRNAs werepredicted. In total, target genes of fourteen conserved and twelve novel barley miRNAfamilies were predicted. Transcription factors, including GAMyb transcription factor, nucleartranscription factor NAC were predicted to be potential targets of barley miRNAs.Furthermore, genes directly involved in protein synthesis, e.g., Serine/threonine kinase-likeprotein and protein synthesis inhibitor were also the targets of barley miRNAs.In summary, our work provided a first systematic analysis of barley transcriptome touncover miRNAs and significantly increases number of novel miRNA in barley. These resultsreveal the miRNA regulation of gene expression and provide theoretical basis for barleygenetic improvement, even for the wheat.