Advertisement

Physiology and Molecular Biology of Plants

, Volume 25, Issue 3, pp 779–786 | Cite as

Expression patterns of cp4-epsps gene in diverse transgenic Saccharum officinarum L. genotypes

  • Muhammad Imran
  • Andre Luiz Barboza
  • Shaheen Asad
  • Zafar M. Khalid
  • Zahid MukhtarEmail author
Research Article
  • 51 Downloads

Abstract

Glyphosate, a functional analogue of phosphoenolpyruvate (PEP), blocks the shikimate pathway by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) through interference with the conversion of (shikimate-3-phosphate) S3P and PEP to 5-enolpyruvylshikimate-3-phosphate (EPSP) and subsequently leads to plant death. This metabolic pathway possesses great potential to be used for development of herbicide resistant transgenic crops and here in this study, we wanted to check the expression potential of CP4-EPSPS gene in various sugarcane genotypes. A synthetic version of CP4-EPSPS gene synthesized commercially, cloned in pGreen0029 vector, was transformed into regenerable embryogenic calli of three different sugarcane cultivars HSF-240, S2003US-778 and S2003US-114 using biolistic gene transfer approach for comparative transcriptional studies. Transgenic lines screened by PCR analysis were subjected to Southern hybridization for checking transgene integration patterns. All the tested lines were found to contain multiple (3–6) insert copies. Putative transgenic plants produced the CP4-EPSPS protein which was detected using immunoblot analysis. The CP4-EPSPS transcript expression detected by qRT-PCR was found to vary from genotype to genotype and is being reported first time. In vitro glyphosate assay showed that transformed plants were conferring herbicide tolerance. It is concluded that different cultivars of sugarcane give variable expression of the same transgene and reasons for this phenomenon needs to be investigated.

Keywords

Codon optimization Sugarcane Genotype Glyphosate qRT-PCR Transcripts 

Notes

References

  1. Adamczyk J Jr, Meredith W Jr (2004) 1260501. Genetic basis for variability of Cry1Ac expression among commercial transgenic Bacillus thuringiensis (Bt) cotton cultivars in the United States. J Cotton Sci 8:433–440Google Scholar
  2. Adamczyk JJ, Sumerford DV (2001) Potential factors impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard® cotton. J Insect Sci 1(13):1–6CrossRefGoogle Scholar
  3. Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327CrossRefGoogle Scholar
  4. Bower R, Elliott AR, Potier BA, Birch RG (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breeding 2:239–249CrossRefGoogle Scholar
  5. Butterfield M, D’hont A, Berding N (2001) The sugarcane genome: a synthesis of current understanding, and lessons for breeding and biotechnology. In: Proc S Afr Sug Technol Ass. Citeseer, pp 1–5Google Scholar
  6. Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lu W, Liu Y, Wang J, Lin M (2012) A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE 7:e38718CrossRefGoogle Scholar
  7. Chou T-C, Moyle RL (2014) Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. BMC Plant Biol 14:92CrossRefGoogle Scholar
  8. Dill GM (2005) Glyphosate-resistant crops: history, status and future. Pest Manag Sci 61:219–224CrossRefGoogle Scholar
  9. Dong H, Li W (2007) Variability of endotoxin expression in Bt transgenic cotton. J Agron Crop Sci 193:21–29CrossRefGoogle Scholar
  10. Finn TE, Wang L, Smolilo D, Smith NA, White R, Chaudhury A (2011) Transgene expression and transgene-induced silencing in diploid and autotetraploid Arabidopsis. Genetics 187:409–423CrossRefGoogle Scholar
  11. Finnegan E, Llewellyn D, Fitt G (1998) What’s happening to the expression of the insect protection in field-grown Ingard® cotton. In: The ninth Australian cotton conference proceedings, pp 291–297Google Scholar
  12. Fu H, Dooner HK (2002) Intraspecific violation of genetic colinearity and its implications in maize. Proc Natl Acad Sci USA 99:9573–9578CrossRefGoogle Scholar
  13. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353CrossRefGoogle Scholar
  14. Jackson MA, Anderson DJ, Birch RG (2013) Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants. Transgenic Res 22:143–151CrossRefGoogle Scholar
  15. Jiang N, Bao Z, Zhang X, Eddy SR, Wessler SR (2004) Pack-MULE transposable elements mediate gene evolution in plants. Nature 431:569–573CrossRefGoogle Scholar
  16. Kinkema M, Geijskes J, Palupe A, Shand K, Coleman HD, Brinin A, Williams B, Sainz M, Dale JL (2014) Improved molecular tools for sugar cane biotechnology. Plant Mol Biol 84:497–508CrossRefGoogle Scholar
  17. Lai J, Ma J, Swigoňová Z, Ramakrishna W, Linton E, Llaca V, Tanyolac B, Park Y-J, Jeong O-Y, Bennetzen JL (2004) Gene loss and movement in the maize genome. Genome Res 14:1924–1931CrossRefGoogle Scholar
  18. Noguera A, Enrique R, Perera MF, Ostengo S, Racedo J, Costilla D, Zossi S, Cuenya MI, Filippone MP, Welin B (2015) Genetic characterization and field evaluation to recover parental phenotype in transgenic sugarcane: a step toward commercial release. Mol Breeding 35:1–15CrossRefGoogle Scholar
  19. Pettigrew W, Adamczyk J (2006) Nitrogen fertility and planting date effects on lint yield and Cry1Ac (Bt) endotoxin production. Agron J 98:691–697CrossRefGoogle Scholar
  20. Pollegioni L, Schonbrunn E, Siehl D (2011) Molecular basis of glyphosate resistance: different approaches through protein engineering. FEBS J 278:2753–2766CrossRefGoogle Scholar
  21. Sachs E, Benedict J, Stelly D, Taylor J, Altman D, Berberich S (1998) Expression and segregation of genes encoding CryIA insecticidal proteins in cotton. Crop Sci 38:1–11CrossRefGoogle Scholar
  22. Tzin V, Galili G (2010) The biosynthetic pathways for shikimate and aromatic amino acids in Arabidopsis thaliana. Arabidopsis Book 8:e0132CrossRefGoogle Scholar
  23. Vickers J, Grof C, Bonnett G, Jackson P, Morgan T (2005) Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane clones in the field. Crop Pasture Sci 56:57–68CrossRefGoogle Scholar
  24. Welch M, Villalobos A, Gustafsson C, Minshull J (2011) 3 Designing genes for successful protein expression. Method Enzymol 498:43CrossRefGoogle Scholar
  25. Wessler SR (2001) Plant transposable elements. A hard act to follow. Plant Physiol 125:149–151CrossRefGoogle Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  1. 1.Agricultural Biotechnology DivisionNational Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
  2. 2.Pakistan Institute of Engineering and Applied SciencesNilore, IslamabadPakistan
  3. 3.Laboratory of Genomics and Molecular Biology, Department of Biological SciencesEscola Superior Agricultura Luiz De Quiroz, University of Sao PauloPiracicabaBrazil
  4. 4.Department of Bioinformatics and BiotechnologyInternational Islamic UniversityIslamabadPakistan

Personalised recommendations