Journal of Applied Phycology

, Volume 31, Issue 2, pp 1175–1183 | Cite as

Small RNA, transcriptome, and degradome sequencing to identify salinity stress responsive miRNAs and target genes in Dunaliella salina

  • Xiangnan Gao
  • Yuting Cong
  • Jinrong Yue
  • Zhenyu Xing
  • Yuan Wang
  • Xiaojie ChaiEmail author


Dunaliella salina is known as the most salinity-tolerant unicellular eukaryote. To explore its molecular response mechanisms to high salinity concentrations, D. salina transcriptomes, small RNA groups and degradomes were analyzed under salinity stress conditions, by high throughput sequencing. A total of 1008 microRNA (miRNA) sequences were identified, including 998 known conserved miRNAs and 10 novel miRNAs. Further analysis of miRNA expression in D. salina under salinity stress found that 49miRNAs showed significant differences in expression. For the first time in D. salina, 745 target genes, regulated by 194 miRNAs, were validated by degradome sequencing. Gene ontology (GO) enrichment analysis and KEGG analysis showed that these miRNA target genes are involved in a variety of molecular biological regulation processes, such as signal transduction, material transport, transcriptional regulation and protein processing. In combination with transcriptome sequencing results, 14 differentially expressed miRNAs and 87 differentially expressed target genes were found to negatively correlate in expression. Further analysis showed that mmu-miR-466, dme-miR-2493, mmu-mir-669h, dre-mir-29a and dme-mir-9388 play an important role in osmoregulation in response to high salinity stress in D. salina. These results enrich existing hypotheses, while providing new insights into the molecular mechanism of salinity tolerance in D. salina.


Differential expression Dunaliella salina High throughput sequencing miRNA Salinity stress 



This work was supported by the National Natural Science Foundation of China (No. 31472260). D. salina cells were provided by the Aquatic Biology Laboratory of Dalian Ocean University, Dalian, China.


  1. Addoquaye C, Miller W, Axtell MJ (2009) CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 25:130–131CrossRefGoogle Scholar
  2. Belmans D, Van Laere A (2010) Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress. Plant Cell Environ 10:185–190Google Scholar
  3. Bočvar DA (2008) Regulation of salt-induced starch degradation in Dunaliella tertiolecta. J Plant Physiol 127:77–96Google Scholar
  4. Borowitzka MA (2013) Dunaliella: biology, production, and markets. In: Richmond A, Hu Q (eds) Handbook of microalgal culture. John Wiley & Sons, Ltd, London, pp 359–368CrossRefGoogle Scholar
  5. Borowitzka MA (2018) The ‘stress’ concept in microalgal biology—homeostasis, acclimation and adaptation. J Appl Phycol.
  6. Chen Z, Jiao XZ, Liu H (1991) Role of the plasma membrane H+-ATPase during the osmoregulation of the alga Dunaliella salina under hypertonic stress. Plant Physiol J 17:333–341Google Scholar
  7. Chen H, Lao YM, Jiang JG (2011) Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina. Sci Total Environ 409:1291–1297CrossRefGoogle Scholar
  8. Degani H, Sussman I, Peschek GA, Avron M (1985) 13C- and 1H-NMR studies of osmoregulation in Dunaliella. Biochim Biophys Acta Mol Cell Res 846:313–323CrossRefGoogle Scholar
  9. Emmer E, Rood RP, Wesolek JH, Cohen ME, Braithwaite RS, Sharp GWG, Murer H, Donowitz M (1989) Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+ /H+ antiporter. J Membr Biol 108:207–215CrossRefGoogle Scholar
  10. Gao P, Bai X, Yang L, Lv D, Li Y, Cai H, Ji W, Guo D, Zhu Y (2010) Over-expression of Osa-MIR396c decreases salt and alkali stress tolerance. Planta 231:991–1001CrossRefGoogle Scholar
  11. Gao P, Bai X, Yang L, Lv D, Pan X, Li Y, Cai H, Ji W, Chen Q, Zhu Y (2011) Osa-MIR393: a salinity- and alkaline stress-related microRNA gene. Mol Biol Rep 38:237–242CrossRefGoogle Scholar
  12. Goyal A (2007a) Osmoregulation in Dunaliella, part I: effects of osmotic stress on photosynthesis, dark respiration and glycerol metabolism in Dunaliella tertiolecta and its salt-sensitive mutant (HL 25/8). Plant Physiol Biochem 45:696–704CrossRefGoogle Scholar
  13. Goyal A (2007b) Osmoregulation in Dunaliella, part II: photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem 45:705–710CrossRefGoogle Scholar
  14. Han X, Yin H, Song X, Zhang Y, Liu M, Sang J, Jiang J, Li J, Zhuo R (2016) Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation. Plant Biotechnol J 14:1470–1483CrossRefGoogle Scholar
  15. Harmon AC (2000) CDPKs-a kinase for every Ca2+ signal? Trends Plant Sci 5:154–159CrossRefGoogle Scholar
  16. He Q, Qiao D, Bai L, Zhang Q, Yang W, Li Q, Cao Y (2007) Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina. J Plant Physiol 164:214–220CrossRefGoogle Scholar
  17. Issa AA (1996) The role of calcium in the stress response of the halotolerant green alga Dunaliella bardawil Ben-Amotz et Avron. Phyton 36:295–302Google Scholar
  18. Jagadeeswaran G, Saini A, Sunkar R (2009) Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta 229:1009–1014CrossRefGoogle Scholar
  19. Jia Y, Xue L, Liu H, Li J (2009) Characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the halotolerant alga Dunaliella salina and inhibition of its expression by RNAi. Curr Microbiol 58:426–431CrossRefGoogle Scholar
  20. Jones AK, Sattelle DB (2008) The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans. Invertebr Neurosci 8:41–47CrossRefGoogle Scholar
  21. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53CrossRefGoogle Scholar
  22. Katz A, Pick U (2001) Plasma membrane electron transport coupled to Na+ extrusion in the halotolerant alga Dunaliella. Biochim Biophys Acta Bioenerg 1504:423–431CrossRefGoogle Scholar
  23. Katz A, Waridel P, Shevchenko A, Pick U (2007) Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis. Mol Cell Proteomics 6:1459–1472CrossRefGoogle Scholar
  24. Li Z, Meng X, Liu C, Yu L, Chen X (2006) Effects of osmotic stress on intracellular glycerol content and enzyme activity in Dunaliella salina. Chinese Bull Bot 23:145–151Google Scholar
  25. Li B, Qin Y, Hui D, Yin W, Xia X (2011) Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica. J Exp Bot 62:3765–3779CrossRefGoogle Scholar
  26. Li SP, Dong HX, Yang G, Wu Y, Su SZ, Shan XH, Liu HK, Han JY, Liu JB, Yuan YP (2016) Identification of microRNAs involved in chilling response of maize by high-throughput sequencing. Biol Plant 60:251–260CrossRefGoogle Scholar
  27. Liska AJ, Shevchenko A, Pick U, Katz A (2004) Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics. Plant Physiol 136:2806–2817CrossRefGoogle Scholar
  28. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836–843CrossRefGoogle Scholar
  29. Means AR (1994) Calcium, calmodulin and cell cycle regulation. FEBS Lett 347:1–4CrossRefGoogle Scholar
  30. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Maréchal-Drouard L (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250CrossRefGoogle Scholar
  31. Mukherjee A (2015) Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii. Mol Biol 49:742–754Google Scholar
  32. Pantaleo V, Szittya G, Moxon S, Miozzi L, Moulton V, Dalmay T, Burgyan J (2010) Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. Plant J 62:960–976Google Scholar
  33. Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A 102:3691–3696CrossRefGoogle Scholar
  34. Pick U, Avron M (1992) Modulation of Na+/H+ antiporter activity by extreme pH and salt in the halotolerant alga Dunaliella salina. Plant Physiol 100:1224–1229CrossRefGoogle Scholar
  35. Popova LG, Shumkova GA, Andreev IM, Balnokin YV (2005) Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima. FEBS Lett 57922:5002CrossRefGoogle Scholar
  36. Qin Z, Chen J, Jin L, Duns GJ, Ouyang P (2015) Differential expression of miRNAs under salt stress in Spartina alterniflora leaf tissues. J Nanosci Nanotechnol 15:1554–1561CrossRefGoogle Scholar
  37. Saito H, Posas F (2012) Response to hyperosmotic stress. Genetics 192:289–318CrossRefGoogle Scholar
  38. Sun XF, Huang F, Liang X, Zhang FW, Yang WG, Bai LH, Qiao DR, Cao Y (2007) Expression of GPD gene from Dunaliella salina treated with different stress and glycerol synthesis of the cells. J Sichuan Univ 44:433–438Google Scholar
  39. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019CrossRefGoogle Scholar
  40. Walne PR (1966) Experiments in the large scale culture of the larvae of Ostrea edulis. Fish Investig 2:1–53Google Scholar
  41. Wang Y, Li L, Tang S, Liu J, Zhang H, Zhi H, Jia G, Diao X (2016) Combined small RNA and degradome sequencing to identify miRNAs and their targets in response to drought in foxtail millet. BMC Genet 17:57CrossRefGoogle Scholar
  42. Weiss M, Pick U (1990) Transient Na+ flux following hyperosmotic shock in the halotolerant alga Dunaliella salina : a response to intracellular pH changes. J Plant Physiol 136:429–438CrossRefGoogle Scholar
  43. Yu Y, Wu G, Yuan H, Cheng L, Zhao D, Huang W, Zhang S, Zhang L, Chen H, Zhang J (2016) Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses. BMC Plant Biol 16:124CrossRefGoogle Scholar
  44. Zhang XL (2013) Cloning and bioinformatic analysis of CDPK gene of Dunaliella salina. J Nuclear Agric Sci 27:418–424Google Scholar
  45. Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang XJ, Qi Y (2007) A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev 21:1190–1203CrossRefGoogle Scholar
  46. Zhao B, Ge L, Liang R, Li W, Ruan K, Lin H, Jin Y (2009) Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Biol 10:1–10CrossRefGoogle Scholar
  47. Zhao LN, Gong WF, Chen XW, Chen DF (2013) Characterization of genes and enzymes in Dunaliella salina involved in glycerol metabolism in response to salt changes. Phycol Res 61:37–45CrossRefGoogle Scholar
  48. Zhao R, Ng DHP, Fang L, Chow YYS, Yuan KL (2015) MAPK in Dunaliella tertiolecta regulates glycerol production in response to osmotic shock. Eur J Phycol 18:243–248Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Xiangnan Gao
    • 1
  • Yuting Cong
    • 1
  • Jinrong Yue
    • 1
  • Zhenyu Xing
    • 1
  • Yuan Wang
    • 1
  • Xiaojie Chai
    • 1
    Email author
  1. 1.Key Laboratory of Hydrobiology in Liaoning ProvinceDalian Ocean UniversityDalianChina

Personalised recommendations