Advertisement

Vegetos

, Volume 32, Issue 4, pp 600–608 | Cite as

De novo transcriptome sequencing of Monodopsis subterranea CCALA 830 and identification of genes involved in the biosynthesis of eicosapentanoic acid and triacylglycerol

  • Shivangi Shah
  • Dinabandhu Sahoo
  • Rohit Nandan Shukla
  • Girish MishraEmail author
Research Articles
  • 17 Downloads

Abstract

Monodopsis subterranea, a unicellular yellow-green freshwater microalga, is widely known for its ability to produce high amount of therapeutically beneficial ω-3 polyunsaturated fatty acid eicosapentanoic acid (EPA; C20:5Δ5,8,11,14,17). Currently, no genomic information is available on M. subterranea despite its nutraceutical and commercial applications. Analysis on fatty acid methyl esters from M. subterranea strain CCALA 830 demonstrated accumulation of 28% EPA. In order to obtain better understanding of EPA biosynthesis and to identify genes involved in the process of lipid metabolism and accumulation in this alga, de novo transcriptome sequencing and assembly was performed using Illumina Hiseq 2000 sequencing. A total of 35,954 transcripts were obtained through final transcriptome assembly with an average transcript length of 769.36 bp. BLAST similarity searches for assembled transcripts were performed followed by annotation using Gene ontology and Kyoto Encyclopedia of Genes and Genomes orthology identifiers. Transcripts involved in lipid biosynthesis including various fatty acid desaturases and elongases involved in PUFAs biosynthesis were identified during the study. In addition, sequences for several transcription factors and a number of simple sequence repeats were also ascertained which can be used as powerful genetic markers for further genetic analysis. This study would provide a useful resource for future research on M. subterranea genome.

Keywords

Monodopsis subterranea Transcriptome Illumina HiSeq 2000 Fatty acid biosynthesis TAG EPA 

Notes

Acknowledgements

Authors acknowledge financial support from the Department of Biotechnology, Ministry of Science and Technology, India (BT/PR6027/AGII/106/859/2012). SS is thankful for INSPIRE fellowship (IF140077) from the Department of Science and Technology, India.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

42535_2019_61_MOESM1_ESM.pdf (40 kb)
Supplementary material 1 (PDF 40 kb)

References

  1. Abdellaoui N, Kim MJ, Choi TJ (2019) Transcriptome analysis of gene expression in Chlorella vulgaris under salt stress. World J Microbiol Biotechnol 35:141.  https://doi.org/10.1007/s11274-019-2718-6 CrossRefPubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Arao T, Sakaki T, Yamada M (1994) Biosynthesis of polyunsaturated lipids in the diatom, Phaeodactylum tricornutum. Phytochemistry 36:629–635.  https://doi.org/10.1016/S0031-9422(00)89787-3 CrossRefGoogle Scholar
  4. Arora S, Mishra G (2019) Biochemical modulation of Monodopsis subterranea (Eustigmatophyceae) by auxin and cytokinin enhances eicosapentaenoic acid productivity. J Appl Phycol.  https://doi.org/10.1007/s10811-019-01844-3 CrossRefGoogle Scholar
  5. Chu F, Cheng J, Zhang X et al (2019) Transcriptome and key gene expression related to carbon metabolism and fatty acid synthesis of Chlorella vulgaris under a nitrogen starvation and phosphorus repletion regime. J Appl Phycol.  https://doi.org/10.1007/s10811-019-01811-y CrossRefGoogle Scholar
  6. Cohen Z (1994) Production potential of eicosapentaenoic acid by Monodus subterraneus. J Am Oil Chem Soc 71:941–945.  https://doi.org/10.1007/BF02542258 CrossRefGoogle Scholar
  7. Conesa A, Götz S, García-Gómez JM et al (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676.  https://doi.org/10.1093/bioinformatics/bti610 CrossRefPubMedGoogle Scholar
  8. Corteggiani Carpinelli E, Telatin A, Vitulo N et al (2014) Chromosome scale genome assembly and transcriptome profiling of Nannochloropsis gaditana in nitrogen depletion. Mol Plant 7:323–335.  https://doi.org/10.1093/mp/sst120 CrossRefPubMedGoogle Scholar
  9. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. Genome Res 8:186–194.  https://doi.org/10.1101/gr.8.3.186 CrossRefPubMedGoogle Scholar
  10. Fukuda S, Hirasawa E, Takemura T et al (2018) Accelerated triacylglycerol production without growth inhibition by overexpression of a glycerol-3-phosphate acyltransferase in the unicellular red alga Cyanidioschyzon merolae. Sci Rep.  https://doi.org/10.1038/s41598-018-30809-8 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gao C, Wang Y, Shen Y et al (2014) Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes. BMC Genomics.  https://doi.org/10.1186/1471-2164-15-582 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Grabherr MG, Haas BJ, Yassour M et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652.  https://doi.org/10.1038/nbt.1883 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Guarnieri MT, Nag A, Smolinski SL et al (2011) Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS One 6:25851.  https://doi.org/10.1371/journal.pone.0025851 CrossRefGoogle Scholar
  14. Haas BJ, Papanicolaou A, Yassour M et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512.  https://doi.org/10.1038/nprot.2013.084 CrossRefPubMedGoogle Scholar
  15. He M, Song H, Chen W et al (2019) Comparative transcriptome analysis of wild type and an oleaginous mutant strain of Desmodesmus sp. reveals a unique reprogramming of lipid metabolism under high light. J Appl Phycol.  https://doi.org/10.1007/s10811-019-01821-w CrossRefGoogle Scholar
  16. Hibberd DJ (1981) Notes on the taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (synonym Xanthophyceae). Bot J Linn Soc 82:93–119.  https://doi.org/10.1111/j.1095-8339.1981.tb00954.x CrossRefGoogle Scholar
  17. Kaye Y, Grundman O, Leu S et al (2015) Metabolic engineering toward enhanced LC-PUFA biosynthesis in Nannochloropsis oceanica: overexpression of endogenous δ12 desaturase driven by stress-inducible promoter leads to enhanced deposition of polyunsaturated fatty acids in TAG. Algal Res 11:387–398.  https://doi.org/10.1016/j.algal.2015.05.003 CrossRefGoogle Scholar
  18. Khozin-Goldberg I, Didi-Cohen S, Shayakhmetova I, Cohen Z (2002) Biosynthesis of eicosapentaenoic acid (EPA) in the freshwater eustigmatophyte Monodus subterraneus (Eustigmatophyceae). J Phycol 38:745–756.  https://doi.org/10.1046/j.1529-8817.2002.02006.x CrossRefGoogle Scholar
  19. Koid AE, Liu Z, Terrado R et al (2014) Comparative transcriptome analysis of four prymnesiophyte algae. PLoS One.  https://doi.org/10.1371/journal.pone.0097801 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kuntal H, Sharma V, Daniell H (2012) Microsatellite analysis in organelle genomes of Chlorophyta. Bioinformation 8:255–259.  https://doi.org/10.6026/97320630008255 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Lauritano C, De Luca D, Ferrarini A et al (2017) De novo transcriptome of the cosmopolitan dinoflagellate Amphidinium carterae to identify enzymes with biotechnological potential. Sci Rep 7:1–12.  https://doi.org/10.1038/s41598-017-12092-1 CrossRefGoogle Scholar
  22. Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659.  https://doi.org/10.1093/bioinformatics/btl158 CrossRefPubMedGoogle Scholar
  23. Li H, Wang W, Wang Z et al (2016a) De novo transcriptome analysis of carotenoid and polyunsaturated fatty acid metabolism in Rhodomonas sp. J Appl Phycol 28:1649–1656.  https://doi.org/10.1007/s10811-015-0703-5 CrossRefGoogle Scholar
  24. Li L, Zhang G, Wang Q (2016b) De novo transcriptomic analysis of Chlorella sorokiniana reveals differential genes expression in photosynthetic carbon fixation and lipid production. BMC Microbiol.  https://doi.org/10.1186/s12866-016-0839-8 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Liang C, Cao S, Zhang X et al (2013) De novo sequencing and global transcriptome analysis of Nannochloropsis sp. (Eustigmatophyceae) following nitrogen starvation. Bioenergy Res 6:494–505.  https://doi.org/10.1007/s12155-012-9269-0 CrossRefGoogle Scholar
  26. Liu B, Benning C (2013) Lipid metabolism in microalgae distinguishes itself. Curr Opin Biotechnol 24:300–309.  https://doi.org/10.1016/j.copbio.2012.08.008 CrossRefPubMedGoogle Scholar
  27. Liu CP, Lin LP (2005) Morphology and eicosapentaenoic acid production by Monodus subterraneus UTEX 151. Micron 36:545–550.  https://doi.org/10.1016/j.micron.2005.05.001 CrossRefPubMedGoogle Scholar
  28. Lv H, Qu G, Qi X et al (2013) Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation. Genomics 101:229–237.  https://doi.org/10.1016/j.ygeno.2013.01.004 CrossRefPubMedGoogle Scholar
  29. Mansfeldt CB, Richter LV, Ahner BA et al (2016) Use of de novo transcriptome libraries to characterize a novel oleaginous marine Chlorella species during the accumulation of triacylglycerols. PLoS One.  https://doi.org/10.1371/journal.pone.0147527 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Meesapyodsuk D, Qiu X (2012) The front-end desaturase: structure, function, evolution and biotechnological use. Lipids 47:227–237.  https://doi.org/10.1007/s11745-011-3617-2 CrossRefPubMedGoogle Scholar
  31. Merchant SS, Kropat J, Liu B et al (2012) TAG, You’re it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. Curr Opin Biotechnol 23:352–363.  https://doi.org/10.1016/J.COPBIO.2011.12.001 CrossRefPubMedGoogle Scholar
  32. Patel RK, Jain M (2012) NGS QC toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7:30619.  https://doi.org/10.1371/journal.pone.0030619 CrossRefGoogle Scholar
  33. Peng KT, Zheng CN, Xue J et al (2014) Delta 5 fatty acid desaturase upregulates the synthesis of polyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum. J Agric Food Chem 62:8773–8776.  https://doi.org/10.1021/jf5031086 CrossRefPubMedGoogle Scholar
  34. Riaño-Pachón D, Ruzicic S, Dreyer I, Mueller-Roeber B (2007) PlnTFDB: an integrative plant transcription factor database. BMC Bioinform 8:42.  https://doi.org/10.1186/1471-2105-8-42 CrossRefGoogle Scholar
  35. Rismani-yazdi H, Haznedaroglu BZ, Bibby K, Peccia J (2011) Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: pathway description and gene discovery for production of next-generation biofuels. BMC Genomics 12:148.  https://doi.org/10.1186/1471-2164-12-148 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Rismani-Yazdi H, Haznedaroglu BZ, Hsin C, Peccia J (2012) Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation. Biotechnol Biofuels 5:74.  https://doi.org/10.1186/1754-6834-5-74 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Santos LM, Leedale GF (1995) Some notes on the ultrastructure of small azoosporic members of the algal class Eustigmatophyceae. Nov Hedwigia 60:219–225Google Scholar
  38. Sato A, Matsumura R, Hoshino N et al (2014) Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii. Front Plant Sci.  https://doi.org/10.3389/fpls.2014.00444 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schroeder A, Mueller O, Stocker S et al (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol.  https://doi.org/10.1186/1471-2199-7-3 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Tautz D, Renz M (1984) Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res 12:4127–4138.  https://doi.org/10.1093/nar/12.10.4127 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106:411–422.  https://doi.org/10.1007/s00122-002-1031-0 CrossRefPubMedGoogle Scholar
  42. Thiriet-Rupert S, Carrier G, Chénais B et al (2016) Transcription factors in microalgae: genome-wide prediction and comparative analysis. BMC Genomics.  https://doi.org/10.1186/s12864-016-2610-9 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Torre S, Tattini M, Brunetti C et al (2016) De Novo assembly and comparative transcriptome analyses of red and green morphs of sweet basil grown in full sunlight. PLoS One 11:1–19.  https://doi.org/10.1371/journal.pone.0160370 CrossRefGoogle Scholar
  44. Vieler A, Wu G, Tsai C-H et al (2012) Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8:1003064.  https://doi.org/10.1371/journal.pgen.1003064 CrossRefGoogle Scholar
  45. Wang W, Li H, Lin X et al (2017) Identification and characterization of miRNAs involved in adventitious branches formation of Gracilaria lichenoides in vitro. J Appl Phycol 29:607–615.  https://doi.org/10.1007/s10811-016-0930-4 CrossRefGoogle Scholar
  46. Wang X, Dong H-P, Wei W et al (2018) Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum. Biotechnol Biofuels 11:318.  https://doi.org/10.1186/s13068-018-1317-3 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Yang H, Mao YX, Kong FN et al (2011) Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology. Chin Sci Bull 56:2119–2130.  https://doi.org/10.1007/s11434-011-4546-4 CrossRefGoogle Scholar
  48. Ye J, Fang L, Zheng H et al (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:293–297.  https://doi.org/10.1093/nar/gkl031 CrossRefGoogle Scholar
  49. Yu WL, Ansari W, Schoepp NG et al (2011) Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 10:91CrossRefGoogle Scholar
  50. Yu M, Yang S, Lin X (2016) De-novo assembly and characterization of Chlorella minutissima UTEX2341 transcriptome by paired-end sequencing and the identification of genes related to the biosynthesis of lipids for biodiesel. Mar Genomics 25:69–74.  https://doi.org/10.1016/j.margen.2015.11.005 CrossRefPubMedGoogle Scholar
  51. Zheng M, Tian J, Yang G et al (2013) Transcriptome sequencing, annotation and expression analysis of Nannochloropsis sp. at different growth phases. Gene 523:117–121.  https://doi.org/10.1016/j.gene.2013.04.005 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Plant Research 2019

Authors and Affiliations

  1. 1.Department of BotanyUniversity of DelhiDelhiIndia
  2. 2.Institute of Bioresources and Sustainable DevelopmentImphalIndia
  3. 3.Bionivid Technology Private LimitedBengaluruIndia

Personalised recommendations