Abstract
We aim to identify conserved and dehydration responsive microRNAs (miRNAs) in Hordeum vulgare (barley). A total of 28 new barley miRNAs belonging to 18 distinct miRNA families were identified. Detailed nucleotide analyses revealed that barley pre-miRNAs are in the range of 46–114 nucleotides with average of 77.14. Using 28 newly detected miRNAs as queries, 445 potential target mRNAs were predicted. The predicted miRNAs were differentially expressed and some of them behaved similarly in leaf and root tissues upon stress treatment. Hvu-MIR156, Hvu-MIR166, Hvu-MIR171, and Hvu-MIR408 were detected as dehydration stress-responsive barley miRNAs. To discover target transcripts of barley miRNAs a modified 5′ RLM-RACE was performed and seven cleaved miRNA transcripts were retrieved from drought stressed leaf samples. In silico analysis indicated 15 potential EST targets. Measurement of expression levels showed a positive correlation between levels of miRNA expression and suppression of their target mRNA transcripts in dehydration-stress-treated barley.
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Abbreviations
- ΔG:
-
Folding free energies
- AGO1:
-
Argonaute-1
- ARF:
-
Auxin response transcription factor
- DCL1:
-
Dicer-like 1
- GSS:
-
Genome survey sequence
- miRNA:
-
Micro-RNA
- RT:
-
Real time
- EST:
-
Expressed sequence tag
- EREBPs:
-
Ethylene-responsive element binding proteins
- MFE:
-
Minimal folding free energy
- MFEI:
-
Minimal folding free energy index
- miRNA*:
-
miRNA star strand
- NCBI:
-
National center for biotechnology information
- nt:
-
Nucleotide(s)
- pre-miRNA:
-
MicroRNA precursor
- pri-miRNAs:
-
MicroRNA primary
- RISC:
-
RNA-induced silencing complex
- 5′ RLM-RACE:
-
RNA ligase-mediated 5′ rapid amplification of cDNA ends
- qRT-PCR:
-
Quantitative real-time PCR
- SBP:
-
Squamosa promoter binding proteins
- SCL6:
-
Scarecrow-like transcription factor 6
- Ser:
-
Serine
- SPL:
-
SBP-like proteins
- Thr:
-
Threonine
- ORF:
-
Open reading frame
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Melda Kantar and Turgay Unver should be regarded as joint first authors.
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Supplementary Table 1
Pre-miRNA sequences of predicted barley miRNAs (DOC 66 kb)
Supplementary Table 2
Computationally identified target ESTs for H. vulgare miRNAs (DOC 45 kb)
Supplementary Table 3
List of primers used for quantification and validation of computer-based H. vulgare miRNAs (DOC 38 kb)
Supplementary Table 4
In silico found miRNA targets that were intended to be amplified in RLM-RACE reactions. The green part indicates in silico miRNA cleavage site, yellow parts indicate reverse primers (DOC 25 kb)
Supplementary Table 5
miRNA cleaved mRNA sequences retrieved with RLM-RACE experiments (DOC 32 kb)
Supplementary Table 6
Mean quantification values and corresponding standard deviations of triplicates for miRNA and target real-time PCRs (DOC 90 kb)
Supplementary Figure 1
Sequence alignment of RACE sequences with computationally proposed Hvu-miRNAs (DOC 27 kb)
Supplementary Figure 2
Sequence alignment of RACE sequences with known miRNAs from different plant species. * indicates reverse complimentary miRNA (DOC 26 kb)
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Kantar, M., Unver, T. & Budak, H. Regulation of barley miRNAs upon dehydration stress correlated with target gene expression. Funct Integr Genomics 10, 493–507 (2010). https://doi.org/10.1007/s10142-010-0181-4
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DOI: https://doi.org/10.1007/s10142-010-0181-4