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Identification of novel stress-induced microRNAs and their targets in Camelina sativa using computational approach

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Abstract

MicroRNAs (miRNAs) are a class of small non-coding, single-stranded RNA sequences that regulate gene expression at the post-transcriptional level and also reported to function in stress responses, but their role has not been studied in Camelina (Camelina sativa L.), an emerging oil crop. In this study, we predicted conserved as well as putative novel miRNAs from a Camelina drought stress cDNA library using comprehensive genomic approaches. Based on the sequence homology, we predicted 145 miRNAs, of which 61 were conserved, and 84 putative novel miRNAs were found to belong to 26 and 72 different miRNA families, respectively. In silico expression analysis indicated that 20 miRNAs were really expressed in Camelina genome, and several of them have tissue-specific expression character. We found that the 60 putative novel miRNA families target 117 genes. Most of the miRNA targets were predicted to genes including that regulate stress response, transcription factors, and fatty acid and lipid metabolism-related genes. Expression patterns of 6 randomly selected miRNAs under drought stress were validated by real-time quantitative polymerase chain reaction analysis. Coordinated expression changes between 6 randomly selected miRNAs and their target genes, suggested that the predicted miRNAs could be drought-responsive and that they would likely be directly involved in stress regulatory networks of Camelina. These results indicate that, in C. sativa, under drought stress, a large number of new miRNAs could be discovered, and the predicted stress-responsive miRNAs and their target transcripts will serve as valuable resources for future studies.

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Acknowledgments

This research was supported by Bio-industry Technology Development Program (No. 312033-5), iPET (Korea Insitute of Planning and Evaluation for Technology in Agriculture, Food and Rural Affairs) and Radiation Technology R&D program through the National Research Foundation of Korea funded by the ministry of Science, ICT & Future Planing (NRF-2013M2A2A6043621).

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Authors and Affiliations

  1. Department of Horticulture, Chungnam National University, Daejeon, 305-764, South Korea

    Saminathan Subburaj & Geung-Joo Lee

  2. Department of Food Science and Technology, Sejong University, Seoul, Korea

    Augustine Yonghwi Kim

  3. Department of Bioresource Engineering, Sejong University, Seoul, Korea

    Sanghyeob Lee

  4. Department of Molecular Biology, Sejong University, Seoul, Korea

    Kyung-Nam Kim

  5. Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea

    Mi Chung Suh

  6. Breeding Institute, Asia Seed Co. Ltd., Icheon, Gyeonggi, Korea

    Gi-Jun Kim

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  1. Saminathan Subburaj
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  2. Augustine Yonghwi Kim
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  4. Kyung-Nam Kim
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11816_2016_395_MOESM1_ESM.docx

Procedure of potential Camelina sativa miRNA gene search by identifying homologs of previously known plant miRNAs (DOCX 13 kb)

11816_2016_395_MOESM2_ESM.pdf

Predicted hairpin secondary structures of the 84 putative novel miRNAs identified in this study. Mature miRNA sequences are highlighted in green. The lengths of the accurate miRNA precursors may be slightly longer than that presented here (PDF 1079 kb)

11816_2016_395_MOESM3_ESM.tif

miRNA mir11736–mir3698 cluster in Camelina EST CaSativa1SL019889t001. a Schematic diagram of organization of the cluster. b EST sequence containing miRNAs encoded within the cluster. Underlined sequences indicate the pre-miRNAs; bold red- and green-colored nucleotides represent the mature miRNAs. c The predicted hairpin structures of mir11736 and mir3698 (TIFF 46083 kb)

11816_2016_395_MOESM4_ESM.xlsx

Conserved miRNAs identified by homolog search and secondary structure in the present study. mir: mature miRNA (XLSX 19 kb)

11816_2016_395_MOESM5_ESM.xlsx

Putative novel miRNAs identified by homolog search and secondary structure in the present study. ML: mature sequence length; MS: mature miRNA sequence arm side; LP: length of pre-miRNAs; NM: number of nucleotide mismatches (XLSX 30 kb)

11816_2016_395_MOESM6_ESM.xlsx

In silico blast expression analysis of novel miRNA genes in different tissues and seed developmental stages from the Camelina transcriptome data (Poudel et al. 2015). Number in the columns indicates number of miRNA hits during BLAST as well as expression in that particular tissue (leaves, buds, and seeds). Absence of number describes miRNA with no BLAST hit or absence of expression. On the right side panel, heatmap depicting the tissue-specific transcript accumulation of miRNAs in various tissues which is produced using the number of miRNA hits during BLAST. A gradient color bar scale represents the transcript accumulation level of high (red) or low (green) (XLSX 23 kb)

11816_2016_395_MOESM7_ESM.docx

List of primer sequences used for qRT-PCR analyses in the present study. miRNA-specific forward primers in combination with universal reverse primer from Mir-X™ miRNA First-Strand synthesis kit (Clontech, USA) were used (DOCX 12 kb)

11816_2016_395_MOESM8_ESM.xlsx

Potential targets of putative novel identified Camelina sativa miRNAs and similar Arabidopsis thaliana genes (XLSX 32 kb)

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Subburaj, S., Kim, A.Y., Lee, S. et al. Identification of novel stress-induced microRNAs and their targets in Camelina sativa using computational approach. Plant Biotechnol Rep 10, 155–169 (2016). https://doi.org/10.1007/s11816-016-0395-6

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