Identification and characterization of miRNAome in tobacco (Nicotiana tabacum) by deep sequencing combined with microarray

Tobacco is one of the most important economic and agricultural crops worldwide. miRNAs have been increasingly acknowledged for their important roles in different biological processes of tobacco. However, few miRNAs have been identified so far in tobacco impeding the development of new tobacco strains with better properties. In this study, high-throughput sequencing technology was employed to identify novel tobacco miRNAs. A total of 84 potential miRNAs were obtained in tobacco, including 33 conserved and 51 novel miRNAs. Tissue-specific and topping-related miRNAs were identified. A tobacco miRNA microarray was also constructed to investigate miRNA expression patterns in different tissues, and their expression patterns were further validated by qRT-PCR and Northern Blot. Finally, the potential targets of these miRNAs were predicted based on a sequence homology search. Thus, in the current study, we have performed the comprehensive analysis of tobacco miRNAs, including their identification, expression pattern and target prediction. Our study opens a new avenue for further elucidation for their roles underlying the regulation of diversity of physiological processes.     

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setariaitalica)

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method.     

Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are an important gene regulator, controlling almost all biological and metabolic processes, in both plants and animals. In this study, we investigated the effect of drought and salinity stress on the expression of miRNAs and their targets in cotton (Gossypium hirsutum L.). Our results show that the expression change of miRNAs and their targets were dose-dependent and tissue-dependent under salinity and drought conditions. The expression of miRNAs in leaf was down-regulated under higher salinity stress while shows variable patterns in other conditions. The highest fold-changes of miRNAs were miR398 in roots with 28.9 fold down-regulation under 0.25% NaCl treatment and miR395 in leaves with 7.6 fold down-regulation under 1% PEG treatment. The highest up-regulation of miRNA targets was AST in roots with 4.7 fold-change under 2.5% PEG and the gene with highest down-regulation was CUC1 in leaves with 25.6 fold-change under 0.25% NaCl treatment. Among seven miRNA-target pairs we studied, five pairs, miR156–SPL2, miR162–DCL1, miR159–TCP3, miR395–APS1 and miR396–GRF1, show significant regulation relationship in roots and leaves under salinity stress concentration.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

Identification and characterization of microRNAs and their target genes in Brassica oleracea

The microRNAs are a new class of small non-coding endogenous RNAs with lengths of approximately ~21 nt. MicroRNAs perform their biological function via the degradation of the target mRNAs or by inhibiting protein translation. Until recently, only limited numbers of miRNAs were identified in Brassica oleracea, a vegetable widely cultivated around the world. In present study, 193 potential miRNA candidates were identified from 17 expressed sequence tag (ESTs) and 152 genome survey sequences (GSSs) in B. oleracea. These miRNA candidates were classified into 70 families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156 and miR158 families. Of these, 36 miRNA families are firstly found in Brassica species. Around 1393 B. oleracea genes were predicted as candidate targets of 175 miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using quantitative real-time polymerase chain reaction (qRT-PCR) and 5' RLM-RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the 818, 514, and 265 target genes are involved in molecular functions, biological processes, and cellular component respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis suggests that these miRNAs might regulate 186 metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen.     

Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia

Hypoxia represents a major physiological challenge for prawn culture, and the hepatopancreas plays an important role in these processes. Here, we applied high-throughput sequencing technology to detect the gene expression profile of the hepatopancreas in Macrobrachium nipponense in response to hypoxia for 3 h and hypoxia for 24 h. Gene expression profiling identified 1925 genes that were significantly up- or down-regulated by dissolved oxygen availability. Functional categorization of the differentially expressed genes revealed that oxygen transport, electron transport chain, reactive oxygen species generation/scavenging, and immune response were the differentially regulated processes occurring during environmental hypoxia. Finally, quantitative real-time polymerase chain reaction using six genes independently verified the tag-mapped results. Immunohistochemistry analysis revealed, for the first time, hemocyanin protein expression as significant hypoxia-specific signature in prawns, which opens the way for in depth molecular studies of hypoxia exposure. The analysis of changes in hepatic gene expression in oriental river prawn provides a preliminary basis for a better understanding of the molecular response to hypoxia exposures.     

Exploring the interaction between small RNAs and R genes during Brachypodium response to Fusarium culmorum infection

The present study aims to investigate small RNA interactions with putative disease response genes in the model grass species Brachypodium distachyon. The fungal pathogen Fusarium culmorum (Fusarium herein) and phytohormone salicylic acid treatment were used to induce the disease response in Brachypodium. Initially, 121 different putative disease response genes were identified using bioinformatic and homology based approaches. Computational prediction was used to identify 33 candidate new miRNA coding sequences, of which 9 were verified by analysis of small RNA sequence libraries. Putative Brachypodium miRNA target sites were identified in the disease response genes, and a subset of which were screened for expression and possible miRNA interactions in 5 different Brachypodium lines infected with Fusarium. An NBS-LRR family gene, 1g34430, was polymorphic among the lines, forming two major genotypes, one of which has its miRNA target sites deleted, resulting in altered gene expression during infection. There were siRNAs putatively involved in regulation of this gene, indicating a role of small RNAs in the B. distachyon disease response.     

Molecular characterisation of TNF,AIF, dermatopontin and VAMP genes of the flat oyster Ostrea edulis and analysis of their modulation by diseases

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes.