RNA-Seq and transcriptome analysis of nitrogen-deprivation responsive genes in Dunaliella salina TG strain

  • Hexin LvEmail author
  • Qiao-e Wang
  • Bingbing Qi
  • Jiatong He
  • Shiru Jia


Nitrogen is a critical macronutrient for plant growth and development, and Dunaliella salina has various physiological responses to nitrogen deprivation, such as accumulating large amounts of carotenoids in its chloroplasts. However, little is known about the underlying global molecular response of D. salina to nitrogen deprivation. Herein, we used the Illumina platform to interrogate which genes of D. salina respond to nitrogen deprivation. The RNA-seq libraries of D. salina TG cultured under nitrogen-replete and -depleted conditions for 7 days generated 87,160,704 valid reads with an average length of 124.94 bp. De novo assembly produced 73,329 transcripts with an average length of 801 bp and 47,283 unigenes with an average length of 675 bp. 5803 unigenes were assigned to 50 gene ontology (GO) terms belonging to three main categories. A total of 6016 unigenes with 671 enzyme commission numbers were assigned to 251 predicted KEGG metabolic pathways, and 6296 unigenes were categorized into 25 KOG classifications. A total of 27,306 CDS were predicted from the 47,283 unigenes. Analysis of transcription levels revealed that 2380 genes were upregulated and 747 were downregulated during nitrogen deprivation. These nitrogen-deprivation responsive genes and differentially expressed transcriptional regulator genes were identified by GO classification. The transcriptome data may serve as a reference for further analysis of gene expression and functional genomics studies, and will facilitate the study of carotenoid biosynthesis and nitrogen metabolism in chlorophytes at the molecular level.


Dunaliella salina Nitrogen deprivation Transcriptome RNA-seq Transcription regulator 



This work is supported by the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University and the National Natural Science Foundation of China (No. 31401029).

Author contributions

SJ and HL conceived and designed the project. HL analyzed the data and wrote the paper. BQ performed the cultures materials preparation and qPCR analysis. QW revised the paper and JH provided bioinformatic analysis tools. All authors have read and approved the final manuscript.

Supplementary material

40626_2019_138_MOESM1_ESM.xlsx (4.8 mb)
Supplementary material 1 (XLSX 4909 kb) Supplement Table 1. Statistics of Gene Annotations by Different Databases.
40626_2019_138_MOESM2_ESM.xlsx (360 kb)
Supplementary material 2 (XLSX 359 kb) Supplement Table 2. KEGG Pathway Classifications of Unigenes.
40626_2019_138_MOESM3_ESM.xlsx (218 kb)
Supplementary material 3 (XLSX 218 kb) Supplement Table 3. Differential Expressions of Unigenes of Cells under CM and -N.
40626_2019_138_MOESM4_ESM.xlsx (82 kb)
Supplementary material 4 (XLSX 82 kb) Supplement Table 4. Clusters of DEGs by Hierarchal Analysis.
40626_2019_138_MOESM5_ESM.xlsx (20 kb)
Supplementary material 5 (XLSX 19 kb) Supplement Table 5. Pathway Classifications of DEGs by KEGG. S gene number: Numbers of significantly changed genes in a given KEGG pathway. B gene number: Numbers of annotated genes in a given KEGG pathway.
40626_2019_138_MOESM6_ESM.xlsx (12 kb)
Supplementary material 6 (XLSX 12 kb) Supplement Table 6. Transcription regulators Classified by WEGO analysis.


  1. Bairoch A, Boeckmann B (1992) The SWISS-PROT protein sequence data bank. Nucleic Acids Res 20(Suppl):2019–2022CrossRefGoogle Scholar
  2. Blaby IK, Glaesener AG, Mettler T, Fitz-Gibbon ST, Gallaher SD, Liu B, Boyle NR, Kropat J, Stitt M, Johnson S, Benning C, Pellegrini M, Casero D, Merchant SS (2013) Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant. Plant Cell 25:4305–4323CrossRefGoogle Scholar
  3. Burge C, Karlin S (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268:78–94CrossRefGoogle Scholar
  4. Cai H, Lu Y, Xie W, Zhu T, Lian X (2012) Transcriptome response to nitrogen starvation in rice. J Biosciences 37:731–747CrossRefGoogle Scholar
  5. Coesel SN, Baumgartner AC, Teles LM, Ramos AA, Henriques NM, Cancela L, Varela JC (2008) Nutrient limitation is the main regulatory factor for carotenoid accumulation and for Psy and Pds steady state transcript levels in Dunaliella salina Chlorophyta exposed to high light and salt stress. Mar Biotechnol 10(5):602–611CrossRefGoogle Scholar
  6. Consortium GO (2006) The gene ontology GO Project in 2006. Nucleic Acids Res 34:D322CrossRefGoogle Scholar
  7. de Lomana ALG, Schäuble S, Valenzuela J, Imam S, Carter W, Bilgin DD, Yohn CB, Turkarslan S, Reiss DJ, Orellana MV (2015) Transcriptional program for nitrogen starvation-induced lipid accumulation in Chlamydomonas reinhardtii. Biotechnol Biofuels 8:207CrossRefGoogle Scholar
  8. Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210CrossRefGoogle Scholar
  9. Emanuelsson O, Nielsen H, Von HG (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978CrossRefGoogle Scholar
  10. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M (2014) The Pfam protein families database. Nucleic Acids Res 42:D222–D230CrossRefGoogle Scholar
  11. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652CrossRefGoogle Scholar
  12. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484CrossRefGoogle Scholar
  13. Krapp A, Daniel-Vedele F (2011) Arabidopsis roots and shoots show distinct temporal adaptation patterns toward nitrogen starvation. Plant Physiol 157:1255–1282CrossRefGoogle Scholar
  14. Lamers PP, Janssen M, De Vos RC, Bino RJ, Wijffels RH (2008) Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends Biotechnol 26:631–638CrossRefGoogle Scholar
  15. Lamers PP, Janssen M, De Vos RC, Bino RJ, Wijffels RH (2012) Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. J Biotechnol 162:21–27CrossRefGoogle Scholar
  16. Lang JD, Hendricks WPD (2018) Identification of driver mutations in rare cancers: the role of SMARCA4 in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). Methods Mol Biol 1706:367–379CrossRefGoogle Scholar
  17. Lers A, Biener Y, Zamir A (1990) Photoinduction of massive β-carotene accumulation by the alga Dunaliella bardawil kinetics and dependence on gene activation. Plant Physiol 93:389–395CrossRefGoogle Scholar
  18. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12:323CrossRefGoogle Scholar
  19. Liang C, Cao S, Zhang X, Zhu B, Su Z, Dong X, Guang X, Ye N (2013) De novo sequencing and global transcriptome analysis of Nannochloropsis sp. Eustigmatophyceae following nitrogen starvation. Bioenerg Res 6:494–505CrossRefGoogle Scholar
  20. Lv H, Qu G, Qi X, Lu L, Tian C, Ma Y (2013) Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation. Genomics 101(4):229–237CrossRefGoogle Scholar
  21. Lv H, Cui X, Wahid F, Xia F, Zhong C, Jia S (2016a) Analysis of the physiological and molecular responses of Dunaliella salina to macronutrient deprivation. PLoS ONE 11:e0152226CrossRefGoogle Scholar
  22. Lv H, Cui X, Wang S, Jia S (2016b) Metabolic profiling of Dunaliella salina shifting cultivation conditions to nitrogen deprivation. Metab Open Acess 6:2153Google Scholar
  23. Lv H, Cui X, Tan Z, Jia S (2017) Analysis of metabolic responses of Dunaliella salina to phosphorus deprivation. J Appl Phycol 29:1251–1260CrossRefGoogle Scholar
  24. Mamedov TG, Moellering ER, Chollet R (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas reinhardtii. Plant J 42:832–843CrossRefGoogle Scholar
  25. Miller R, Wu G, Deshpande RR, Vieler A, Gärtner K, Li X, Moellering ER, Zäuner S, Cornish AJ, Liu B, Bullard B, Sears BB, Kuo MH, Hegg EL, Shachar-Hill Y, Shiu SH, Benning C (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154:1737–1752CrossRefGoogle Scholar
  26. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628CrossRefGoogle Scholar
  27. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline syst 1:1–14CrossRefGoogle Scholar
  28. Polle JEW, Barry K, Cushman J, Schmutz J, Tran D, Hathwaik LT, Yim WC, Jenkins J, McKie-Krisberg Z, Prochnik S, Lindquist E, Dockter RB, Adam C, Molina H, Bunkenborg J, Jin E, Buchheim M, Magnuson J (2017) Draft nuclear genome sequence of the halophilic and beta-carotene-accumulating green alga. Genome Announc. 5(43):e01105–e01117CrossRefGoogle Scholar
  29. Pruitt KD, Tatiana T, Maglott DR (2005) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 33:D501–D504CrossRefGoogle Scholar
  30. Ramos A, Coesel S, Marques A, Rodrigues M, Baumgartner A, Noronha J, Rauter A, Brenig B, Varela J (2008) Isolation and characterization of a stress-inducible Dunaliella salina Lcy-beta gene encoding a functional lycopene beta-cyclase. Appl Microbiol Biot 79:819–828CrossRefGoogle Scholar
  31. Ramos AA, Marques AR, Rodrigues M, Henriques N, Baumgartner A, Castilho R, Brenig B, Varela JC (2009) Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salina. J Plant Physiol 166:968–977CrossRefGoogle Scholar
  32. Ramos AA, Polle J, Tran D, Cushman JC, Jin E, Varela JC (2011) The unicellular green alga Dunaliella salina Teod. as a model for abiotic stress tolerance: genetic advances and future perspectives. Algae 26:3–20CrossRefGoogle Scholar
  33. Salguero A, de la Morena B, Vigara J, Vega JM, Vilchez C, León R (2003) Carotenoids as protective response against oxidative damage in Dunaliella bardawil. Biomol Eng 20:249–253CrossRefGoogle Scholar
  34. Seo J, Gordishdressman H, Hoffman EP (2006) An interactive power analysis tool for microarray hypothesis testing and generation. Bioinformatics 22:808CrossRefGoogle Scholar
  35. Simionato D, Block MA, La Rocca N, Jouhet J, Maréchal E, Finazzi G, Morosinotto T (2013) The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus. Eukaryot Cell 12:665–676CrossRefGoogle Scholar
  36. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 28:33CrossRefGoogle Scholar
  37. von Wirén N, Lauter FR, Ninnemann O, Gillissen B, Walch-Liu P, Engels C, Jost W, Frommer WB (2000) Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato. Plant J. 21(2):167–175CrossRefGoogle Scholar
  38. Weng L-C, Pasaribu B, Lin I-P, Tsai C-H, Chen C-S, Jiang P-L (2014) Nitrogen deprivation induces lipid droplet accumulation and alters fatty acid metabolism in symbiotic dinoflagellates isolated from Aiptasia pulchella. Sci Rep 4:5777CrossRefGoogle Scholar
  39. Wickham H (2009) ggplot2: Elegant Graphics for Data Analysis. Springer Publishing Company, Incorporated.
  40. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2016) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45:D200–D203CrossRefGoogle Scholar
  41. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293CrossRefGoogle Scholar
  42. Zhang X-N, Qu Z-C, Wan Y-Z, Zhang H-W, Shen D-L (2002) Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA in Dunaliella salina (chlorophyta) under hyperosmotic shock. Plant Mol Biol Rep 20:49–57CrossRefGoogle Scholar
  43. Zschiedrich CP, Keidel V, Szurmant H (2016) Molecular mechanisms of two-component signal transduction. J Mol Biol 428:3752–3775CrossRefGoogle Scholar

Copyright information

© Brazilian Society of Plant Physiology 2019

Authors and Affiliations

  1. 1.Key Laboratory of Industrial Fermentation MicrobiologyMinistry of Education (Tianjin University of Science & Technology)TianjinChina
  2. 2.Beijing Key Lab of Plant Resource Research and DevelopmentBeijing Technology and Business UniversityBeijingChina

Personalised recommendations