Abstract
Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1–4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bhullar S, Chakravarthy S, Pental D, Burma PK (2009) Analysis of promoter activity in transgenic plants by normalizing expression with a reference gene: anomalies due to the influence of the test promoter on the reference promoter. J Biosci 34(6):953–962
Cakmak I, Yazici A, Tutus Y, Ozturk L (2009) Glyphosate reduced seed and leaf concentrations of calcium, manganese, magnesium, and iron in non-glyphosate resistant soybean. Eur J Agron 31:114–119
Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lin M (2012) A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants. PLoS ONE 7:e38718
Chhapekar S, Raghavendrarao S, Pavan G, Ramakrishna C, Singh VK, Phanindra MLV, Kumar PA (2015) Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate. Plant Cell Rep 34:721–731
Dill GM (2005) Glyphosate-resistant crops: history, status and future. Pest Manag Sci 61:219–224
Dun B, Wang X, Lu W, Chen M, Zhang W, Ping S, Lin M (2014) Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes. Crop J 2:164–169
Garg R, Chevala VN, Shankar R, Jain M (2015) Divergent DNA methylation patterns associated with gene expression in rice cultivars with contrasting drought and salinity stress response. Sci Rep 5:14922
Gianessi LP (2013) The increasing importance of herbicides in worldwide crop production. Pest Manag Sci 69:1099–1105
Guo X, Han H, Zhang J, Wang H (2003) The viral resistance of transgenic tobacco plants containing untranslatable PVYN coat protein gene. Shi Yan Sheng Wu Xue Bao 36:176–184
Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A, Hoffman H (1984) Inheritance of functional genes in plants. Science 223:496–498
Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95–97
Liu Y, Cao G, Chen R, Zhang S, RenY LuW, Wang G (2015) Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene. Transgenic Res 24:753–763
Main GD, Reynolds S, Gartland JS (1995) Electroporation protocols for Agrobacterium. Methods Mol Biol 44:405–412
Sammons RD, Gaines TA (2014) Glyphosate resistance: state of knowledge. Pest Manag Sci 70:1367–1377
Te Z, Lin CY, Shen ZC (2011) Development of transgenic glyphosate-resistant rice with G6 gene encoding 5-enolpyruvylshikimate-3-phosphate synthase. Agr Sci China 10:1307–1312
Tizaoui K, Kchouk ME (2012) Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco. Genet Mol Biol 35:640–649
Wang J, Zuo K, Lu W, Zhu Y, Ye C, Lin M, Tang K (2014) Over-expression of the Gr5 aroA gene from glyphosate-contaminated soil confers high tolerance to glyphosate in tobacco. Mol Breeding 33:197–208
Ye GN, Hajdukiewicz PT, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Imran, M., Asad, S., Barboza, A.L. et al. Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide. Physiol Mol Biol Plants 23, 453–460 (2017). https://doi.org/10.1007/s12298-017-0424-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12298-017-0424-0