Functional & Integrative Genomics

, Volume 10, Issue 4, pp 493–507

Regulation of barley miRNAs upon dehydration stress correlated with target gene expression

Original Paper

DOI: 10.1007/s10142-010-0181-4

Cite this article as:
Kantar, M., Unver, T. & Budak, H. Funct Integr Genomics (2010) 10: 493. doi:10.1007/s10142-010-0181-4

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.

Keywords

Hordeum vulgaremicroRNAStem-loop hairpin structureExpressed sequence tagTarget mRNAqRT-PCRDehydration stress5′ RLM-RACE

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

Supplementary material

10142_2010_181_MOESM1_ESM.doc (66 kb)
Supplementary Table 1Pre-miRNA sequences of predicted barley miRNAs (DOC 66 kb)
10142_2010_181_MOESM2_ESM.doc (46 kb)
Supplementary Table 2Computationally identified target ESTs for H. vulgare miRNAs (DOC 45 kb)
10142_2010_181_MOESM3_ESM.doc (38 kb)
Supplementary Table 3List of primers used for quantification and validation of computer-based H. vulgare miRNAs (DOC 38 kb)
10142_2010_181_MOESM4_ESM.doc (26 kb)
Supplementary Table 4In 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)
10142_2010_181_MOESM5_ESM.doc (32 kb)
Supplementary Table 5miRNA cleaved mRNA sequences retrieved with RLM-RACE experiments (DOC 32 kb)
10142_2010_181_MOESM6_ESM.doc (90 kb)
Supplementary Table 6Mean quantification values and corresponding standard deviations of triplicates for miRNA and target real-time PCRs (DOC 90 kb)
10142_2010_181_MOESM7_ESM.doc (27 kb)
Supplementary Figure 1Sequence alignment of RACE sequences with computationally proposed Hvu-miRNAs (DOC 27 kb)
10142_2010_181_MOESM8_ESM.doc (26 kb)
Supplementary Figure 2Sequence alignment of RACE sequences with known miRNAs from different plant species. * indicates reverse complimentary miRNA (DOC 26 kb)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural SciencesSabanci UniversityIstanbulTurkey