Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress
- First Online:
- Cite this article as:
- Paul, S., Kundu, A. & Pal, A. Plant Cell Tiss Organ Cult (2011) 105: 233. doi:10.1007/s11240-010-9857-7
- 674 Views
MicroRNAs (miRNAs) are 20–24 nucleotide long non-coding RNAs known to play important regulatory roles during plant development, organ morphogenesis, and stress responses by controlling gene expression. Although Vigna unguiculata (cowpea) is an economically important salt sensitive member of legumes, very little is known about the conserved miRNAs and their expression profile during salinity stress in this plant. In the present study using comparative genomic approach and following a set of strict filtering criteria we have identified 18 conserved V. unguiculata miRNAs belonging to 16 distinct miRNA families. Using these potential miRNA sequences 15 potential target genes were predicted and all of them were identified as transcription factors. Seven of these predicted V. unguiculata miRNAs were experimentally validated in the root tissues and found to be up-regulated during salt stress as revealed by quantitative real time PCR (qRT-PCR). Perfectly cleaved Auxin response factor (ARF), the target transcript of V. unguiculata miR160 was detected successfully by modified 5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method.
KeywordsMicroRNASalt stressVigna unguiculata (cowpea)miRNA targetsqRT-PCR5′ modified RLM-RACE
Expressed sequence tag
Genomic survey sequence
RNA induced silencing complex
Minimum folding free energy
Minimum folding free energy index
RNA ligase-mediated rapid amplification of cDNA ends
Quantitative real time PCR
Dana Farber Cancer Institute
Squamosa promoter binding protein
Squamosa promoter binding protein-like protein
Auxin response factor
CCAAT-binding transcription factor
Nuclear factor Y
Small nuclear RNA
Vigna unguiculata (L.) Walp. (cowpea) is one of the important leguminous crops in the semi-arid tropics covering Asia, Africa, southern Europe, Central and South America (Singh et al. 1997). Cowpea has excellent nutritional qualities containing 24–26 percent protein and well balanced essential amino acid composition with high amounts of leucine, lysine and methionine (Bressani 1985). It is also capable of enhancing soil fertility through biological nitrogen fixation (Martins et al. 2003). However, legume production has been highly affected by salinity stress (Bayuelo-Jimenez et al. 2002; Wang et al. 2003; Chen et al. 2007), the most severe abiotic stress; which can limit growth and development of plants (Munns 1993). MicroRNAs have a great role in gene regulation but very limited information is available to date about V. unguiculata microRNAs and their expression pattern during stress.
MicroRNAs (miRNAs) are a class of small, non-coding, evolutionarily conserved RNAs with about 20–24 nucleotides in length (Bonnet et al. 2004). They play crucial roles in post-transcriptional gene regulation by complementing the target mRNAs and causing transcriptional repression or target mRNA degradation (Bartel 2004). In plants, RNA polymerase II enzyme transcribes miRNA genes into long primary transcripts (pri-miRNAs; Chen 2005). This pri-miRNAs are subsequently trimmed by ribonuclease III-like Dicer (DCL1) enzyme producing miRNA precursors (pre-miRNAs) with stem-loop (hairpin) structure(s). Subsequently, a second cleavage by DCL1 at the loop region of the hairpin produces a short double-stranded RNA (dsRNA). One of these strand acts as mature miRNA (Kurihara and Watanabe 2004). The mature miRNA gets incorporated into the RNA induced silencing complex (RISC) and guides RISC to complementary mRNA targets for degradation (Lin et al. 2005). Lots of investigations indicated that majority of characterized miRNAs are involved in plant development (Chen 2004), signaling (Yoshikawa et al. 2005) and organ morphogenesis (Kidner and Martienssen 2005). Recently, it has been reported that miRNAs are also hypersensitive to abiotic or biotic stresses. MicroRNAs possibly regulate gene expression at the post-transcriptional level thus contribute to the stress-induced changes in proteins (Sunkar et al. 2006; Lu et al. 2008; Zhou et al. 2008; Zhang et al. 2008a, b; Ding et al. 2009).
The most common experimental approach to find out novel miRNAs is by direct cloning (Lu et al. 2005; Zhang et al. 2006a), in which small RNAs are first isolated by size fractionation in a denaturing polyacrylaminde gel. Then RNA adapters are ligated to these RNAs at their respective 5′ and 3′ ends. Subsequently, these fragments are reverse transcribed into cDNAs and at last, the first strand cDNAs is amplified by PCR and sequenced (Lu et al. 2005). However, it is very difficult to detect miRNAs that are expressed at low level following this technique. To overcome this limitation, an alternate approach is in vogue, here the novel mature miRNAs and their precursors are first identified by screening the Expressed Sequence Tag (EST)/Genome Survey Sequence (GSS) database of a particular species (Wang et al. 2004; Liang et al. 2007) and then validated by cloning, Northern blotting or quantitative real time PCR (Liang et al. 2007; Feng et al. 2009). Since miRNAs are very much conserved in nature this approach works well, for those species which have sufficient EST/GSS sequences available to predict and validate novel miRNAs (Wang et al. 2004; Liang et al. 2007) that usually cannot be detected by the direct cloning approach due to their low abundance. However recent advances in high-throughput or next generation sequencing strategies can also identify low abundance or tissue specific miRNAs (Fahlgren et al. 2007; Wei et al. 2009) but it requires high technical expertise.
Materials and methods
Reference set of miRNAs
To identify potential V. unguiculata miRNAs, a set of plant miRNAs has been compared with the V. unguiculata EST and GSS database. The set of miRNAs and their precursors used, downloaded from miRBase (version 14.0, September, 2009; http://microrna.sanger.ac.uk/sequences/index.shtml) consisted of 402 known mature miRNA sequences including Arabidopsis thaliana (199) and 4 members of Fabaceae family like Glycine max (85), Lotus japonicus (2), Medicago truncatula (108) and Phaseolus vulgaris (8). Experimentally validated mature miRNA sequences of these plants were mainly used in this computational prediction study.
EST and GSS source for V. unguiculata
V. unguiculata ESTs and GSSs were obtained from NCBI Genbank (http://www.ncbi.nlm.nih.gov/). At present a total number of 2,41,606 sequences including 1,87,660 ESTs and 54194 GSSs of V. unguiculata are deposited in NCBI Genbank.
Prediction of potential V. unguiculata miRNAs and their precursors (pre-miRNAs)
Prediction of potential targets
To determine the potential targets of predicted miRNAs in V. unguiculata, we used BLASTn algorithm against Cowpea (Version 1.0) Dana Farber Cancer Institute (DFCI) Gene Indices (http://compbio.dfci.harvard.edu/tgi/). Previous study has shown that most known miRNAs bind to the protein coding region of their mRNA targets with perfect or near perfect sequence complementarity and degrade or repress the target mRNA (Wang et al. 2004). We followed previously employed criteria to predict potential mRNA targets of given plant miRNAs (Zhang et al. 2007; Yin et al. 2008) and they are: (a) no more than 4 mismatches were allowed at complementary sites between miRNA sequence and potential mRNA targets, (b) no mismatch between positions 10 and 11 (assumed to be cleavage site), no more than 1 mismatch between positions 1 and 9, no more than 2 mismatches at other positions and no gaps between miRNA and target mRNA at the complementary sites were allowed.
Plant materials and stress treatment
Vigna unguiculata (Cv. Local) seeds after surface sterilization in 70% ethanol for 2 min were rinsed twice in deionized water and then placed on water-moistened filter papers in sterile petridishes for germination at 28 ± 1°C and 70% of relative humidity (RH). Seeds germinated in 1 day were transplanted into earthen pots filled with sterile and moist vermiculite and grown in a greenhouse at 25–27°C. Uniform seedlings with two leaves were transferred into a sterile conical flask containing 100 ml of Hoagland nutrient solution (pH 6.5; Hoagland and Arnon 1950). Plants were grown in the described hydroponic system in a greenhouse with controlled environmental conditions (25°C, 70% humidity and natural illumination). The nutrient solution was replaced every 3 days and the conical flask was shaken daily to ensure aeration in the root system. When the first trifoliate leaf became flat, plants were transferred into either a nutrient solution containing 200 mM NaCl for salt stress treatment or the nutrient solution without NaCl as mock. The roots of ten stressed plants were harvested at 12 h after salt stress treatment; the roots of untreated control (mock) were also harvested at the same time point. Harvested roots were immediately frozen in liquid N2 and stored in −80°C freezer until used for RNA isolation.
Small RNA and total RNA isolation
Small RNA and total RNA were isolated from roots of mock and stress treated plants using mirPremier microRNA Isolation Kit (Sigma-Aldrich) and RNeasy Mini Kit (Qiagen) respectively, according to the manufacturer’s instructions. The quality and quantity of isolated RNA samples were measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, USA) and stored at −20°C.
Polyadenylation and cDNA synthesis
Small RNA (1 μg) isolated both from roots of mock and stress-treated plants were polyadenylated and reverse transcribed at 37°C for 1 h in 10 μl reaction mixture using Mir-X miRNA First-Strand Synthesis kit (Clontech) following manufacture’s instructions. The reaction mixture contains 1× mRQ Buffer and 1 μl of mRQ enzyme mix provided with the kit, after 1 h the reaction was terminated at 85°C for 5 min and finally the volume was made up to 100 μl with deionized water.
Detection and cloning of predicted miRNAs
To detect the predicted miRNAs, the obtained cDNA from the previous step was amplified by GeneAmp PCR system 2400 (Perkin Elmer) using entire predicted miRNA sequence as sense primer and mRQ 3′ primer provided with Mir-X miRNA qRT-PCR SYBR kit (Clontech) as antisense primer. The resulting PCR products were checked on 4% agarose gel with EtBr staining and then the gel slice containing desired fragments around 70 bp were excised and eluted using Qiaquick gel elution kit (Qiagen). Finally, The DNA fragments were cloned in pJET1.2 cloning vector provided with CloneJET PCR Cloning Kit (Fermentas) and sequenced (ABI Prism 3130xl DNA sequencer).
Analysis of mature miRNAs expression pattern during salt stress by quantitative real-time PCR (qRT-PCR)
Analysis of the expression pattern of detected mature miRNAs during salt stress was performed by iQ5 quantitative real-time PCR system and iQ5 Optical system software (Bio-Rad) using Mir-X miRNA qRT-PCR SYBR kit (Clontech) following manufacturer’s instructions. Briefly, 25 μl PCR reaction mixtures were prepared and each contained 1× SYBR Advantage Premix, 1× ROX dye, 0.2 μM each of sense and antisense primers as indicated above and 2 μl of the first strand cDNA. The reactions were incubated in a 96 well plate at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s. This cycle was followed by a melting curve analysis ranging from 56 to 95°C, with temperature increasing steps of 0.5°C every 10 s. Melting curves for each amplicon were observed carefully to confirm the specificity of the primers used. Relative expression levels for each sample were obtained using the “comparative Ct method” (Schmittgen and Livak 2008). The threshold cycle (Ct) value obtained after each reaction was normalized to the Ct value of U6 snRNA (U6 snRNA primer was provided with the kit) whose expression was consistent across the conditions. All reactions were conducted in triplicate.
miRNA target validation by 5′ RLM-RACE
To experimentally validate the cleavage sites of computationally predicted targets of the detected miRNAs, we have used a modified version of 5′ RLM-RACE approach (Wei et al. 2009; Arenas-Huertero et al. 2009). One μg of total RNA isolated from stress treated root was subjected to a 5′ RACE reaction using FirstChoice RLM-RACE kit (Ambion) omitting calf intestine alkaline phosphatase and tobacco acid pyrophosphatase treatments. For each Tentative Contig (TC) two gene specific reverse primers (5′ RACE gene specific outer and inner primer) were designed. The PCR reaction and cycling conditions were setup following the manufacture’s protocol. Annealing temperatures were adjusted for specific primers. Finally, the nested PCR products were cloned into pJET1.2 cloning vector (Fermentas) and sequenced (ABI Prism 3130xl DNA sequencer).
Results and discussion
Prediction of potential V. unguiculata miRNAs and their precursors (pre-miRNAs)
Identified conserved miRNAs in Vigna unguiculata
A + U (%)
Prediction of potential miRNA targets using bioinformatics tool
Potential targets of identified Vigna unguiculata miRNAs
Squamosa promoter-binding protein (SBP)
TCP family transcription factor
Auxin response factor (ARF)
CCAAT-binding transcription factor (CBF)
Nuclear transcription factor Y (NFY)
APETALA2 protein (AP2)
Basic blue copper protein/Plantacyanin
TC9874, TC9017, TC16529, TC1781
Experimental validation of predicted miRNAs in V. unguiculata and measurement of their expression level by quantitative real-time PCR (qRT-PCR)
It has been assumed that plant development and plant adaptation to stress are two different but closely related processes. During conditions of stress, expression of most plant miRNAs essential for plant development and morphogenesis is altered (Sunkar et al. 2007). These altered levels of miRNAs indirectly inhibit development and morphogenesis by regulating expression patterns of their target genes. Subsequently, this indirectly allows plants to mobilize essential resources toward adaptive responses to stress (Sunkar et al. 2007).
Identification of V. unguiculata miRNA targets with 5′ RLM-RACE
To verify cleavage sites of computationally predicted targets of validated miRNAs, a modified 5′ RLM-RACE was performed using total RNA isolated from salt stress-treated root tissues of V. unguiculata. Presence of miRNA directed cleavage products using primers specific to the target transcripts were checked. Finally, one of the target transcripts, TC986 for vun-miR160, was validated successfully where the cleavage occurred opposite to the 10th position from the 5′ end of the miRNA vun-miR160. This is in accordance with the previous reports in other plant species, where majority of the miRNA-RISC complex cleaved their potential target transcripts specifically at that position (Arenas-Huertero et al. 2009; Chen et al. 2009). The validated target (TC986) of vun-miR160 was classified as transcription factor ARF. In this experiment though several potential target transcripts were computationally predicted, validation of only one target has been possible, that is in agreement with Chen et al. (2009) and Zeng et al. (2009) who also have failed to validate all successfully predicted miRNA targets by RLM-RACE.
In this study, several conserved miRNAs in V. unguiculata were predicted and validated. A detailed study is underway to understand the roles of miRNAs in adaptive responses to salt stress in V. unguiculata.
We are thankful to the Director, Bose Institute for providing the lab facilities; we also thank the Department of Biotechnology, India, for the financial assistance (Sanction no. BT/01/COE/06/03), a SRF to AK and a RA to SP.