Dataset Information

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber.

ABSTRACT: MicroRNAs (miRNAs) are endogenous small RNAs playing an important regulatory function in plant development and stress responses. Among them, some are evolutionally conserved in plant and others are only expressed in certain species, tissue or developmental stages. Cucumber is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, two independent small RNA libraries from cucumber leaves and roots were constructed, respectively, and sequenced with the high-throughput Illumina Solexa system. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified. QRT-PCR analysis revealed that some of the cucumber miRNAs were preferentially expressed in certain tissues. With the recently developed 'high throughput degradome sequencing' approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. Our study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

SUBMITTER: Mao W

PROVIDER: S-EPMC3316546 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber.

Mao Weihua W Li Zeyun Z Xia Xiaojian X Li Yadan Y Yu Jingquan J

PloS one 20120330 3

MicroRNAs (miRNAs) are endogenous small RNAs playing an important regulatory function in plant development and stress responses. Among them, some are evolutionally conserved in plant and others are only expressed in certain species, tissue or developmental stages. Cucumber is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, ...[more]

PMID: 22479356

Similar Datasets

Project description:MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding the molecular mechanisms of drought resistance in mulberry.

Project description:Powdery mildew (PM) is a prevalent disease known to limit cucumber production worldwide. MicroRNAs (miRNAs) are single-stranded molecules that regulate host defense responses through posttranscriptional gene regulation. However, which specific miRNAs are involved and how they regulate cucumber PM resistance remain elusive. A PM-resistant single-segment substitution line, SSSL508-28, was developed previously using marker-assisted backcrossing of the PM-susceptible cucumber inbred D8 line. In this study, we applied small RNA and degradome sequencing to identify PM-responsive miRNAs and their target genes in the D8 and SSSL508-28 lines. The deep sequencing resulted in the identification of 156 known and 147 novel miRNAs. Among them, 32 and six differentially expressed miRNAs (DEMs) were detected in D8 and SSSL508-28, respectively. The positive correlation between DEMs measured by small RNA sequencing and stem-loop quantitative real-time reverse transcription-polymerase chain reaction confirmed the accuracy of the observed miRNA abundances. The 32 DEMs identified in the PM-susceptible D8 were all upregulated, whereas four of the six DEMs identified in the PM-resistant SSSL508-28 were downregulated. Using in silico and degradome sequencing approaches, 517 and 20 target genes were predicted for the D8 and SSSL508-28 DEMs, respectively. Comparison of the DEM expression profiles with the corresponding mRNA expression profiles obtained in a previous study with the same experimental design identified 60 and three target genes in D8 and SSSL508-28, respectively, which exhibited inverse expression patterns with their respective miRNAs. In particular, five DEMs were located in the substituted segment that contained two upregulated DEMs, Csa-miR172c-3p and Csa-miR395a-3p, in D8 and two downregulated DEMs, Csa-miR395d-3p and Csa-miR398b-3p, in SSSL508-28. One gene encoding L-aspartate oxidase, which was targeted by Csa-miR162a, was also located on the same segment and was specifically downregulated in PM-inoculated D8 leaves. Our results will facilitate the future use of miRNAs in breeding cucumber varieties with enhanced resistance to PM.

Project description:MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted.Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response.We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in 'Yingshuang', and 46 up-regulated miRNA and 45 down-regulated miRNAs in 'Baiye 1' in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

Dataset Information

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber.

Publications

A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets