, Volume 248, Issue 3, pp 613–621

Expression analysis of sugarcane shaggy-like kinase (SuSK) gene identified through cDNA subtractive hybridization in sugarcane (Saccharum officinarum L.)

  • Vikas Yadav Patade
  • Archana Neeraj Rai
  • Penna Suprasanna
Short Communication


Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e−108), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling.


Sugarcane SSH Shaggy kinase Transcript expression Osmotic stress RWC 


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Claisse G, Charrier B, Kreis M (2007) The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion. Plant Mol Biol 64:113–124PubMedCrossRefGoogle Scholar
  3. Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov K, Gurskaya N, Sverdlov E, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue specific cDNA probes and libraries. Proc Natl Acad Sci 93:6025–6030PubMedCrossRefGoogle Scholar
  4. Dornelas MC, Lejeune B, Dron M, Kreis M (1998) The Arabidopsis SHAGGY-related protein kinase (ASK) gene family: structure, organization and evolution. Gene 212:249–257PubMedCrossRefGoogle Scholar
  5. Duguid JR, Dinauer MC (1990) Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucleic Acids Res 18:2789–2792PubMedCrossRefGoogle Scholar
  6. Eck RV, Dayhoff MO (1966) Atlas of protein sequence and structure. National Biomedical Research Foundation, Silver SpringsGoogle Scholar
  7. Embi N, Rylatt DB, Cohen P (1980) Glycogen synthase kinase from rabbit skeletal muscle: separation from cyclic AMP dependent protein kinase and phosphorylase kinase. Eur J Biochem 107:519–527PubMedCrossRefGoogle Scholar
  8. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  9. Ferkey DM, Kimelman D (2000) GSK-3: new thoughts on an old enzyme. Dev Biol 225:471–479PubMedCrossRefGoogle Scholar
  10. Hanks SK (1991) Eukaryotic protein kinases. Current Opinion in Structural Biology 1(3):369–383Google Scholar
  11. Henkoff S, Greene EA, Pietrokovski S, Bork P, Attwood PTK, Hood L (1997) Gene families: the taxonomy of protein paralogs and chimeras. Science 278:609–637CrossRefGoogle Scholar
  12. Li J, Nam KH (2002) BIN2 a new brassinosteroid-insensitive locus in Arabidopsis. Science 295:1299–1301PubMedGoogle Scholar
  13. Li H-Y, Huang S-H, Shi Y-S, Song Y-C, Zhao J-R, Wang F-G, Wang T-Y, Li Y (2007) Isolating soil drought-induced genes from maize seedling leaves through suppression subtractive hybridization. Agril Sci China 6:647–651Google Scholar
  14. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971PubMedCrossRefGoogle Scholar
  15. Liu X, Mei Z, Jun D, Keqiang W (2008) Gene expression analysis of germinating rice seeds responding to high hydrostatic pressure. J Plant Physiol 165:1855–1864PubMedCrossRefGoogle Scholar
  16. Menossi MC, Siva-Filho M, Vincentz MA, Van-Sluys GM (2008) Sugarcane functional genomics: gene discovery for agronomic trait development. International J Plant Genomics. doi:10.1155/2008/458732 Google Scholar
  17. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681PubMedCrossRefGoogle Scholar
  18. Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New YorkGoogle Scholar
  19. Nikolakaki E, Coffer PJ, Hemelsoet R, Woodgett JR, Defize LH (1993) Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells. Oncogene 8:833–840PubMedGoogle Scholar
  20. Piao HL, Pih KT, Lim JH, Kang SG, Jin JB, Kim SH, Hwang I (1999) An Arabidopsis GSK3/shaggy-like gene that complements yeast salt stress-sensitive mutants is induced by NaCl and abscisic acid. Plant Physiol 119:1527–1534PubMedCrossRefGoogle Scholar
  21. Piao HL, Lim JH, Kim SJ, Cheong GW, Hwang I (2001) Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis. Plant J 27:305–314PubMedCrossRefGoogle Scholar
  22. Rodrigues FA, de Laia ML, Zingaretti SM (2009) Analysis of gene expression profiles under water stress in tolerant and sensitive sugarcane plants. Plant Sci 176:286–302CrossRefGoogle Scholar
  23. Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologists programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana, Totowa, pp 365–386Google Scholar
  24. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  25. Tichtinsky G, Tavares R, Takvorian A, Schwebel-Dugue N, Twell D, Kreis M (1998) An evolutionary conserved group of plant GSK-3/shaggy-like protein kinase genes preferentially expressed in developing pollen. Biochemica Biophys Acta 1442:261–273Google Scholar
  26. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science 270:484–487PubMedCrossRefGoogle Scholar
  27. Vettore AL, da Silva FR, Kemper EL, Arruda P (2001) The libraries that made SUCEST. Genet Mol Biol 24:1–7CrossRefGoogle Scholar
  28. Wang X-L, He R-F, He G-C (2005) Construction of suppression subtractive hybridization libraries and identification of brown plant hopper-induced genes. J Plant Physiol 162:1254–1262PubMedCrossRefGoogle Scholar
  29. Wua Y, Wanga O, MaaY CC (2005) Isolation and expression analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method. Plant Sci 168:847–853CrossRefGoogle Scholar
  30. Yang GP, Ross DT, Kang WW, Brown PO, Weigel RJ (1999) Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res 27:1517–1523PubMedCrossRefGoogle Scholar
  31. Zouari N, Saad RB, Legavre T, Azaza J, Sabau X, Jaoua M, Masmoudi K, Hassairi A (2007) Identification and sequencing of ESTs from the halophyte grass Aeluropus littoralis. Gene 404:61–69PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Vikas Yadav Patade
    • 1
    • 2
  • Archana Neeraj Rai
    • 1
  • Penna Suprasanna
    • 1
  1. 1.Functional Plant Biology Section, Nuclear Agriculture and Biotechnology DivisionBhabha Atomic Research CentreTrombayIndia
  2. 2.Molecular Biology and Genetic Engineering DivisionDefence Institute of Bio-Energy ResearchHaldwaniIndia

Personalised recommendations