Journal of Applied Phycology

, Volume 31, Issue 1, pp 183–190 | Cite as

cDNA cloning of gs, gogat, and gdh from Haematococcus pluvialis and transcription and enzyme level analysis in different nitrogen concentration

  • Yating Ding
  • Xiaonan ZangEmail author
  • Jiawei Shi
  • Lulu Hou
  • Bangxiang He
  • Manman Dong
  • Xiaomei Cong
  • Xuexue Cao
  • Zhu Liu
  • Xinwei Song
  • Xiaoyun Huang
  • Xuecheng Zhang


Haematococcus pluvialis has attracted attention due to its ability to produce astaxanthin, which has many functions such as antioxidation and anticancer. In order to further understand its nitrogen metabolism, the transcription levels and the enzyme levels of glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the process of nitrogen assimilation were studied under different nitrogen concentrations after cloning their cDNA sequences. The cloned cDNA sequence of gs is 1438 bp, including a 178-bp 5′ untranslatable region (UTR), a 114-bp 3′ UTR, and a 1146-bp open reading frame (ORF), which encodes a protein of 381 amino acids. The gogat had a total cDNA of 4931 bp, including a 17-bp 5′ UTR and a 4914-bp ORF, which encodes a protein of 1637 amino acids. The gdh had a total cDNA of 1529 bp, including 131-bp 3′ UTR and a 1398-bp ORF, which encodes a protein of 465 amino acids. During cultivation the transcription levels of the three genes fluctuated slightly during the first 4 days and then declined significantly. At the fourth day, the highest level of the transcription of the three genes was at the nitrogen concentration of 1000 mg L−1. The enzyme level also fluctuated with culture time, which is positively correlated with the transcription level. This study may lay foundation for optimization of nitrogen concentration for cultivation of H. pluvialis.


Enzyme level Haematococcus pluvialis Nitrogen assimilation Nitrogen concentration Transcription level 


Funding information

This work was supported by the National Natural Science Foundation of China (Grant No. 31472255).

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  1. Feng Z, Qin ZW, Wu T, He MH (2012) Cloning and expression of cytosolic glutamine synthetase (GS1) in Cucumis sativus L. under low nitrogen conditions. Sci Agric Sin 45:3100–3107Google Scholar
  2. Hou LL, Liu F, Zang XN, Zhang XC, He BX, Ding YT, Song XW, Xiao DF, Wang HT (2017) Cloning and transcription analysis of the nitrate reductase gene from Haematococcus pluvialis. Biotechnol Lett 39:1–9CrossRefGoogle Scholar
  3. Huang GC, Tian B (2001) The physiological role of glutamate dehydrogenase in higher plants. Chinese Bulletin of Botany 18:396–401Google Scholar
  4. Huang Q, Yin L (1995) Influence of nitogen sources on glutamine synthetase in wheat seedling. Acta Bot Sin 11:856–862Google Scholar
  5. Kim S, Lee Y, Hwang SJ (2013) Removal of nitrogen and phosphorus by Chlorella sorokiniana cultured heterotrophically in ammonia and nitrate. Int Biodeterior Biodegrad 85:511–516CrossRefGoogle Scholar
  6. Lea PJ, Miflin BJ (2003) Glutamate synthase and the synthesis of glutamate in plants. Plant Physiol Biochem 41:555–564CrossRefGoogle Scholar
  7. Lea PJ, Robinson SA, Stewart GR (1990) The enzymology and metabolism of glutamine, glutamate, and asparagine. In: Stumpf PK, Conn EE (eds) The biochemistry of plants, vol 16. Academic Press, New York, pp 121–159Google Scholar
  8. Li CJ, Lin QH, Zhang CF (2000) Study on ammonium-assimilating enzymes and isozymes in hgiher plants. J Lingling Teachers’ College 21:20–22Google Scholar
  9. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  10. Luo F, Lu YE, Yang M, Xing M (2012) Effects of nitrogen deficiency on nitrogen metabolism and expression of genes related during vegetative growth stage of rice. Journal of Huazhong Agricultural University 31:16–22Google Scholar
  11. Ma LQ, Che CC, Wang LM, Bo YU, Yang G (2017) Regulation of stereochemical composition of poly-gama-glutamic acids produced by Bacillus licheniformis within various Mn2+ concentration. Microbiology China 44:1263–1270Google Scholar
  12. Melooliveira R, Oliveira IC, Coruzzi GM (1996) Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Proc Natl Acad Sci U S A 93:4718–4723CrossRefGoogle Scholar
  13. Miflin BJ, Habash DZ (2002) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 53:979–987CrossRefGoogle Scholar
  14. Miflin BJ, Lea PJ (1976) The pathway of nitrogen assimilation in plants. Phytochemistry 15:873–885CrossRefGoogle Scholar
  15. Mo LY, Wu LH, Tao QN (2001) Research advances on GS/GOGAT cycle in higher plants. Plant Nutrition and Fertilizer Science 7:223–231Google Scholar
  16. Ortego J, Bonal R, Muoz A (2004) Accumulation of astaxanthin in flagellated cells of Haematococcus pluvialis—cultural and regulatory aspects. Curr Sci 87:1290–1295Google Scholar
  17. Pouliot Y, Buelna G, Racine C, Noüe J (1989) Culture of cyanobacteria for tertiary wastewater treatment and biomass production. Biol Wastes 29:81–91CrossRefGoogle Scholar
  18. Przytocka-Jsiak M (1976) Growth and survival of Chlorella vulgaris in high concentrations of nitrogen. Acta Microbiol Pol 25:287PubMedGoogle Scholar
  19. Quénet D, Dalal Y (2014) A long non-coding RNA is required for targeting centromeric protein A to the human centromere. Elife 4:24724–24735Google Scholar
  20. Scehley KA, Yamaya T, Oaks A (1992) Compartmentation of nitrogen assimilation in higher plants. Int Rev Cytol 134:85–163CrossRefGoogle Scholar
  21. Schoenbeck MA, Temple SJ, Trepp GB, Blumenthal JM, Samac DA, Gantt JS, Georgina H, Carroll PV (2000) Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with antisense glutamate synthase transgene. J Exp Bot 51:29–39PubMedGoogle Scholar
  22. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  23. Thompson JD, Gibson TJ, Plewniak F (1997) The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  24. Wallsgrove RM, Turner JC, Hall NP, Kendally AC, Bright SWJ (1987) Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis. Plant Physiol 83:155–158CrossRefGoogle Scholar
  25. Wang YH, Wang ZQ, Zhang CF, Zhou ZX, Ma JK, Ou JQ (2004) Effect of nitrate nitrogen on activities of glutamine synthetase and glutamate dehydrogenase during development of cucumber cotyledon. Journal of Wuhan Botanical Research 22:534–538Google Scholar
  26. Wang XC, Zhang HR, Wei YH, Jia XT, Gu MX, Ma XM (2017) Differential expression and assembly mode of glutamine synthetase isoenzymes in different tissues and organs of maize. Acta Agron Sin 43:1410–1414CrossRefGoogle Scholar
  27. Wu YJ, Yang TL, Zhang XQ (2014) The influence of glutamine synthetase inhibitors on nitrogen metabolism of Tobacco leaves during senescence period. Tobacco Science 35:37–42Google Scholar
  28. Yu JL, Zhu ZK, Zhang ZH, Rong XM, Liu Q, Song HX, Guan CY (2014) Effects of enzymes related to nitrogen reuse on nitrogen redistribution and nitrogen use efficiency in Brassica napus. Agric Sci Technol 2:215–218Google Scholar
  29. Zhang GY (2012) Effect of different nitrogen levels treatment on activity and expression of key enzyme of carbon and nitrogen. Ph.D. Thesis. Fujian Agriculture and Forestry University. Fuzhou, Fujian, ChinaGoogle Scholar
  30. Zheng CF, Sun HZ, Tang YW (1990) Effects of isonicotinly hydrazine on the level of nitrate reductase activity in Chlamydomonas reinhardii. Chin Sci Bull 35:423–426Google Scholar
  31. Zhu C, Fan Q, Wang W, Shen C, Meng X, Tang Y, Xu Z, Song R (2014) Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana. Gene 536:407–415CrossRefGoogle Scholar
  32. Zou C, Fan X, Shi R, Zhang F (2007) Effect of ammonium and nitrate nitrogen on the growth and iron nutrition of up-and lowland rice. Journal of China Agricultural University 12:45–49Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Yating Ding
    • 1
  • Xiaonan Zang
    • 1
    Email author
  • Jiawei Shi
    • 1
  • Lulu Hou
    • 1
  • Bangxiang He
    • 1
  • Manman Dong
    • 1
  • Xiaomei Cong
    • 1
  • Xuexue Cao
    • 1
  • Zhu Liu
    • 1
  • Xinwei Song
    • 1
  • Xiaoyun Huang
    • 1
  • Xuecheng Zhang
    • 1
  1. 1.Key Laboratory of Marine Genetics and Breeding, Ministry of Education/ College of Marine Life SciencesOcean University of ChinaQingdaoChina

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