3 Biotech

, 9:130 | Cite as

Functional characterization of the rice root Germin-like protein gene-1 (OsRGLP1) promoter in Nicotiana tabacum

  • Muhammad IlyasEmail author
  • Wasim Akhtar
  • Shazia Rehman
  • Syed Muhammad Saqlan Naqvi
  • Tariq Mahmood
Original Article


Germin (GER) and germin-like protein (GLP) genes play a very important role against various stresses. Promoter analysis provide significant insight into gene’s function and regulation. Presently, upstream region (1228 bp) of the OsRGLP1 gene was functionally characterized via heterologous expression. It was fused with the Glucuronidase (GUS) reporter gene and the expression cassette was used to transform Nicotiana tabacum using Agrobacterium-mediated transformation. Transgenic plants were examined via quantitative real-time PCR (qPCR) to analyze its role in wounding, salinity, drought, abscisic acid (ABA) and circadian rhythm. OsRGLP1 was highly induced by ABA and drought by showing 28- and 25-fold changes in GUS mRNA level respectively as compared to wounding (fourfold change) and salinity (threefold change). However, no activity was observed in circadian rhythm. Histochemically, strong GUS activity was observed in leaf veins, midrib, epidermal hair, stomata guard cells, stem cortex, root hairs, xylem and phloem and at cellular level in cell wall, cytoplasm and its periphery. OsRGLP1 promoter can be used to develop agronomically important transgenic plants in future food program.


Rice germin-like proteins OsRGLP1 Germin Germin-like protein Promoter GUS Stress 



We are thankful to The Higher Education Commission (HEC) of Pakistan for providing financial assistance. Grant no.: 17-5-(2BM1-351)HEC/Sch-ind/2012.

Author contributions

MI and TM conducted experiments while SR, WA and SMSN helped in manuscript writing.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest in the publication.


  1. Bai XG, Long J, Li S, Li KZ, Xu HN (2014) Molecular cloning and characterization of a germin-like protein gene in spinach (SoGLP). J Hortic Sci Biotechnol 89(5):592–598CrossRefGoogle Scholar
  2. Bray EA (2004) Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J Exp Bot 55(407):2331–2341CrossRefGoogle Scholar
  3. Cartharius K et al (2005) MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21(13):2933–2942CrossRefGoogle Scholar
  4. Cheong YH et al (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129(2):661–677CrossRefGoogle Scholar
  5. Curtis IS, Davey MR, Power JB (1995) Leaf disk transformation. Methods Mol Biol 44:59–70PubMedGoogle Scholar
  6. Davidson RM, Reeves PA, Manosalva PM, Leach JE (2009) Germins: a diverse protein family important for crop improvement. Plant Sci 177(6):499–510CrossRefGoogle Scholar
  7. Federico ML, Iñiguez-Luy FL, Skadsen RW, Kaeppler HF (2006) Spatial and temporal divergence of expression in duplicated barley germin-like protein-encoding genes. Genetics 174(1):179–190CrossRefGoogle Scholar
  8. Finn RD et al (2014) Pfam: the protein families’ database. Nucleic Acids Res 42(D1):281–288Google Scholar
  9. Himmelbach A et al (2010) Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack. Plant Cell 22(3):937–952CrossRefGoogle Scholar
  10. Ilyas M, Naqvi SMS, Mahmood T (2016a) In silico analysis of transcription factor binding sites in promoters of germin-like protein genes in rice. Arch Biol Sci 68(4):863–876CrossRefGoogle Scholar
  11. Ilyas M, Rasheed A, Mahmood T (2016b) Functional characterization of germin and germin-like protein genes in various plant species using transgenic approaches. Biotechnol Lett 38(9):1405–1421CrossRefGoogle Scholar
  12. Jefferson RA (1989) The GUS reporter gene system. Nature 342(6251):837–838CrossRefGoogle Scholar
  13. Le Deunff E, Davoine C, Le Dantec C, Billard JP, Huault C (2004) Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths. Plant J 38(3):421–431CrossRefGoogle Scholar
  14. Li Y et al (2013) Cotton GhMPK6a negatively regulates osmotic tolerance and bacterial infection in transgenic Nicotiana benthamiana and plays a pivotal role in development. FEBS J 280(20):5128–5144CrossRefGoogle Scholar
  15. Lim S, Baek W, Lee SC (2014) Identification and functional roles of CaDIN1 in abscisic acid signaling and drought sensitivity. Plant Mol Biol 86(4–5):513–525CrossRefGoogle Scholar
  16. Lu M, Han Y-P, Gao J-G, Wang X-J, Li W-B (2011) Identification and analysis of the germin-like gene family in soybean. BMC Genomics 11:620CrossRefGoogle Scholar
  17. Mahmood T, Hyder MZ, Naqvi SMS (2007) Cloning and sequence analysis of germin-like protein gene-2 promoter from Oryza sativa L. ssp. Indica DNA Seq 18(1):26–32CrossRefGoogle Scholar
  18. Mahmood T, Yasmin T, Haque MI, Naqvi SMS (2013) Characterization of a rice germin-like protein gene promoter. Genet Mol Res 12(121):360–369CrossRefGoogle Scholar
  19. Manosalva PM et al (2009) A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice. Plant Physiol 149:286–296CrossRefGoogle Scholar
  20. Munir F et al (2016) Germin-like protein gene-2 promoter from rice is responsive to fungal pathogens in transgenic potato plants. Funct Integr Genomics 16(1):19–27CrossRefGoogle Scholar
  21. Park CJ et al (2004) Molecular characterization of pepper germin-like protein as the novel PR-16 family of pathogenesis-related proteins isolated during the resistance response to viral and bacterial infection. Planta 219(5):797–806CrossRefGoogle Scholar
  22. Peremarti A et al (2010) Promoter diversity in multigene transformation. Plant Mol Biol 73(4):363–378CrossRefGoogle Scholar
  23. Ruiz-López N et al (2012) Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing. Transgenic Res 21(6):1233–1243CrossRefGoogle Scholar
  24. Sassaki FT, Bravo JP, González ER, Maia IG (2014) Expression pattern and promoter analysis of a Eucalyptus grandis germin-like gene. Plant Mol Biol Rep 33:12–21CrossRefGoogle Scholar
  25. Tabuchi T, Kumon T, Azuma T, Nanmori T, Yasuda T (2003) The expression of a germin-like protein with superoxide dismutase activity in the halophyte Atriplex lentiformis is differentially regulated by wounding and abscisic acid. Physiol Plant 118(4):523–531CrossRefGoogle Scholar
  26. Thaler JS, Bostock RM (2004) Interactions between abscisic-acid-mediated responses and plant resistance to pathogens and insects. Ecology 85(1):48–58CrossRefGoogle Scholar
  27. Thompson EW, Lane BG (1980) Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk mRNA from dry and imbibing embryos. J Biol Chem 255(12):5965–5970PubMedGoogle Scholar
  28. Turgeon R, Wolf S (2009) Phloem transport: cellular pathways and molecular trafficking. Annu Rev Plant Biol 60(1):207–221CrossRefGoogle Scholar
  29. van der Weele CM, Spollen WG, Sharp RE, Baskin TI (2000) Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media. J Exp Bot 51(350):1555–1562CrossRefGoogle Scholar
  30. Wang T et al (2013) Characterization of peanut germin-like proteins, AhGLPs in plant development and defense. PLoS One 8(4):.e61722CrossRefGoogle Scholar
  31. Yang L, Li T, Zhang SC, Gao GL, Yang CW (2013) Characterization of the GLP13 gene promoter in Arabidopsis thaliana. Biol Plant 57(2):231–237CrossRefGoogle Scholar
  32. Yasmin T, Mahmood T, Hyder MZ, Akbar S, Naqvi SMS (2008) Cloning, sequencing and in silico analysis of Germin-like protein gene 1 promoter from Oryza sativa l. Ssp. Indica.  Pak J Bot 40(4):1627–1634Google Scholar
  33. Yin K, Han X, Xu Z, Xue H (2009) Arabidopsis GLP4 is localized to the Golgi and binds auxin in vitro. Acta Biochim Biophys Sin 41(6):478–487CrossRefGoogle Scholar
  34. Zimmermann G, Bäumlein H, Mock HP, Himmelbach A, Schweizer P (2006) The multigene family encoding germin-like proteins of barley. Regulation and function in Basal host resistance. Plant Physiol 142(1):181–192CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Muhammad Ilyas
    • 1
    Email author
  • Wasim Akhtar
    • 4
  • Shazia Rehman
    • 5
  • Syed Muhammad Saqlan Naqvi
    • 3
  • Tariq Mahmood
    • 2
  1. 1.Department of BotanyUniversity of SwabiAnbar SwabiPakistan
  2. 2.Department of Botany, Faculty of Biological sciencesQuaid-i-Azam UniversityIslamabadPakistan
  3. 3.Bacha Khan UniversityCharsaddaPakistan
  4. 4.Department of BotanyMohi-Ud-Din Islamic UniversityAzad Jammu and KashmirPakistan
  5. 5.Department of BotanyUniversity of Gujrat sub campus RawalpindiRawalpindiPakistan

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