Abstract
The small, non-coding RNA species (20–24 nucleotides), including microRNAs (miRNAs) and small interfering RNAs, are key regulators of gene expression in eukaryotic cells. Mostly, miRNAs have been identified from glycophytes under different abiotic stresses. The isolation and identification of miRNAs from halophytes are challenging due to the presence of high salt concentrations. In this study, small RNA cDNA libraries were constructed using an improved method to identify salt-responsive novel miRNAs from Salicornia brachiata. A total of 159 sequences were cloned and analysed. There was no adapter contamination and low-abundance small RNAs were amplified efficiently. Twelve putative miRNAs/pre-miRNAs and a small RNA were identified and confirmed by northern blot. Among the putative miRNAs, nine are novel and three belong to the conserved miRNA families (MIR169g, MIR1433, MIR138b). Northern hybridisation and real-time PCR showed that nine miRNAs (seven novel and two conserved) and one small RNA were highly expressed at 2.0 M NaCl, two novel miRNAs at 1.5 M NaCl, and one conserved miRNA at 1.0 M NaCl. Furthermore, 67 putative target genes of the isolated miRNA were predicted on the basis of sequence homology, of which more than 50 % are associated with abiotic stresses. The target genes of miRNA 169g (Sb-miRNA10), and Sb-miRNA7 (NF-YA transcription factor and cytochrome P450-like TATA box binding protein respectively) were analysed by real-time PCR. The study of miRNAs from an extreme halophyte broadens our understanding of the important role played by miRNAs in plant abiotic stress.
Similar content being viewed by others
References
Ambrose V et al (2003) A uniform system for microRNA annotation. RNA 9:277–279
Barozai MYK, Baloch IA, Din M (2012) Identification of microRNA and their targets in Helianthus. Mol Biol Rep 39:2523–2532
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Brezikov E, Cuppen E, Plasterk RHA (2006) Approaches to microRNA discovery. Nat Genet 38:S2–S7
Chen C et al (2005) Real-time quantification of miRNAs by stem-loop RT-PCR. Nucleic Acid Res 33:e179
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Friedlander MR et al (2009) High-resolution profiling and discovery of planarian small RNAs. Proc Natl Acad Sci USA 106:11546–11551
Gao P, Bai X, Yang L, Lv D, Pan X, Li Y et al (2011) osa-MIR393: a salinity- and alkaline stress-related microRNA gene. Mol Biol Rep 38:237–242
Glazov EA, Cottee PA, Barris WC, Moore RJ, Dalrymple BP, Tizard ML (2008) A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. Genome Res 18:957–964
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Gu S, Kay MA (2010) How do miRNAs mediate translational repression? Silence 7(1):11
Jha B, Agarwal PK, Reddy S, Lal S, Sopory SK, Reddy MK (2009) Identification of salt-induced gene from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis. Genes Genet Syst 84:111–120
Jones-Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520
Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53
Kim VN (2005) Small RNAs: classification, biogenesis, and function. Mol Cells 19:1–15
Kruszka K, Pieczynski M, Windels D, Bielewicz D, Jarmolowski A, Szweykowska-Kulinska Z, Vazquez F (2012) Role of microRNAs and other sRNAs of plants in their changing environments. J Plant Physiol 169:1664–1672
Landgraf P et al (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129:1401–1414
Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory role in Caenorhabditis elegans. Science 294:858–862
Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408
Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2005) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349
Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW (1996) P450 Superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1–42
Pang M, Hanson SF, Zhang J (2012) Cloning and expression studies of novel small RNAs in tetraploid cotton. Plant Mol Biol Rep 30:710–718
Parida AK, Jha B (2010) Salt tolerance mechanism in mangroves: a review. Trees 24:199–217
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626
Sdassi N, Silveri L, Laubier J, Tilly G, Costa J, Layani S, Vilotte JL, Provost FL (2009) Identification and characterization of new miRNAs cloned from normal mouse mammary gland. BMC Genom 10:149
Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019
Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of microRNAs from rice. Plant Cell 17:1397–1411
Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309
Sunkar R, Zhou XF, Zheng Y, Zhang WX, Zhu JK (2008) Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biol 8:25
Taji T, Seki M, Satou M, Sakurai T, Kobayashi M, Ishiyama K, Narusaka Y, Narusaka M, Zhu JK, Shinozaki K (2004) Comparative genomics in salt tolerance between Arabidopsis and Arabidopsis-related halophyte salt cress using Arabidopsis microarray. Plant Physiol 135:1697–1709
Tang S, Wang Y, Li Z, Gui Y, Xiao B, Xie J, Zhu QH, Fan L (2012) Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum). BMC Plant Biol 12:28
Wan LC, Zhang H, Lu S, Zhang L, Qui Z, Zhao Y, Zeng QY, Lin J (2012) Transcriptome-wide identification and characterization of miRNAs from Pinus densata. BMC Genom 13:132
Wang W, Zhao LJ, Tan YX, Ren H, Qi JT (2012) MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma. Carcinogenesis 33:1113–1120
Xue X, Sun J, Zhang Q, Wang Z, Huang Y, Pan W (2008) Identification and characterization of novel microRNAs from Schistosoma japonicum. PLoS ONE 3(12):e4034
Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33
Zang Z et al (2012) Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress. Mol Biol Rep 39:8137–8146
Zhang J, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at global genome level identifies microRNAs involved in cold stress response. BMC Genom 10:449
Zhao CZ et al (2010) Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC Plant Biol 10:3
Zhou J, Zhuo RY, Liu MY, Qiao GR, Jiang J, Li HY, Qiu WM, ZhangXG Lin S (2011) Identification and characterization of novel microRNAs from Populus cathayana Rehd. Plant Mol Biol Rep 29:242–251
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acid Res 31:3406–3415
Acknowledgment
CSIR-CSMCRI Communication No. PRIS-014. The financial support of CSIR (BSC0107–PlaGen), Govt. of India for carrying out this project is gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Singh, D., Jha, B. The isolation and identification of salt-responsive novel microRNAs from Salicornia brachiata, an extreme halophyte. Plant Biotechnol Rep 8, 325–336 (2014). https://doi.org/10.1007/s11816-014-0324-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11816-014-0324-5