Abstract
Phosphinothricin acetyltransferase gene (pat) is an important selectable marker and also a key herbicide trait gene in several commercial products. In maize, the transformation frequency (TF) using pat as a selectable marker is the lowest among the commonly used marker options including epsps, pmi or ppo. Low pat transformation efficiency can become a major bottleneck in our ability to efficiently produce large numbers of events, especially for large molecular stack vectors with multiple trait gene cassettes. The root cause of the lower efficiency of pat in maize is not well understood and it is possible that the causes are multifaceted, including maize genotype, pat marker cassette, trait gene combinations and selection system. In this work we have identified a new variant of pat gene through codon optimization that consistently produced a higher transformation frequency (> 2x) than an old version of the pat gene that has codons optimized for expression in dicot plants. The level of PAT protein in all 16 constructs was also found multifold higher (up to 40 fold) over that of the controls. All of the T0 low copy transgenic plants generated from the 16 different constructs showed excellent tolerance to ammonium glufosinate herbicide spray tests at 4x and 8x recommended field application rates (1x = 595 g active ingredient (ai)/hectare of ammonium glufosinate) in the greenhouse.
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References
Angov E (2011) Codon usage: nature’s roadmap to expression and folding of proteins. Biotechnol J 6:650–659. https://doi.org/10.1002/biot.201000332
Bevan M, Barnes WM, Chilton MD (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385
De Block M et al (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6:2513–2518
Duncan DB (1957) Multiple range tests for correlated and heteroscedastic means. Biometrics 13:164–176. https://doi.org/10.2307/2527799
Ingham DJ, Beer S, Money S, Hansen G (2001) Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31:132–134 (136–140)
Ishida Y (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750
Jayne S, Barbour E, Meyer T (2000) Methods for improving transformation efficiency. US Patent 6,096,947
Jia M, Li Y (2005) The relationship among gene expression, folding free energy and codon usage bias in Escherichia coli. FEBS Lett 579:5333–5337. https://doi.org/10.1016/j.febslet.2005.08.059
Joshi CP, Zhou H, Huang X, Chiang VL (1997) Context sequences of translation initiation codon in plants. Plant Mol Biol 35:993–1001. https://doi.org/10.1023/a:1005816823636
Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
Li X, Volrath SL, Nicholl DB, Chilcott CE, Johnson MA, Ward ER, Law MD (2003) Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize. Plant Physiol 133:736–747. https://doi.org/10.1104/pp.103.026245
Lindsey K (1992) Genetic manipulation of crop plants. J Biotechnol 26:1–28. https://doi.org/10.1016/0168-1656(92)90067-J
Maiti IB, Gowda S, Kiernan J, Ghosh SK, Shepherd RJ (1997) Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains. Transgenic Res 6:143–156
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 53:979–987. https://doi.org/10.1093/jexbot/53.370.979
Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803
Nuccio ML et al (2015) Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions. Nat Biotechnol 33:862–869
Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812
Ow DW, Jacobs JD, Howell SH (1987) Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc Natl Acad Sci USA 84:4870–4874
Perl A, Galili S, Shaul O, Ben-Tzvi I, Galili G (1993) Bacterial dihydrodipicolinate synthase and desensitized aspartate kinase: two novel selectable markers for plant transformation. Bio/Technol. 11:715. https://doi.org/10.1038/nbt0693-715
Plotkin JB, Kudla G (2011) Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet 12:32–42. https://doi.org/10.1038/nrg2899
Que Q et al (2010) Trait stacking in transgenic crops: challenges and opportunities. GM Crops 1:220–229. https://doi.org/10.4161/gmcr.1.4.13439
Que Q et al (2014) Maize transformation technology development for commercial event generation. Front Plant Sci 5:379. https://doi.org/10.3389/fpls.2014.00379
Sivamani E, Starmer JD, Qu R (2009) Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression. Plant Sci 177:549–556. https://doi.org/10.1016/j.plantsci.2009.08.006
Strauch E, Wohlleben W, Puhler A (1988) Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tu494 and its expression in Streptomyces lividans and Escherichia coli. Gene 63:65–74
Tijssen P (1985) Practice and theory of enzyme immunoassays. Elsevier Science, New York
Webster GR, Teh AY, Ma JK (2017) Synthetic gene design-The rationale for codon optimization and implications for molecular pharming in plants. Biotechnol Bioeng 114:492–502
Wenck A et al (2003) Reef-coral proteins as visual, non-destructive reporters for plant transformation. Plant Cell Rep 22:244–251. https://doi.org/10.1007/s00299-003-0690-x
Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Puhler A (1988) Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70:25–37
Wright M et al (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–436. https://doi.org/10.1007/s002990100318
Zhong H et al (2018) Advances in Agrobacterium-mediated maize transformation. Methods Mol Biol 1676:41–59. https://doi.org/10.1007/978-1-4939-7315-6_3
Ziemienowicz A (2001) Plant selectable markers and reporter genes. Acta Physiol Plant 23:363–374. https://doi.org/10.1007/s11738-001-0045-6
Acknowledgements
Help rendered by Rachel Whinna, Pei Su, Ping Wu, Sabrina Patton, Melissa Murray, Jamie McCuiston, Yaping Jiang, Lucy Qin and Yoshimi Barron towards this work are gratefully acknowledged. We are thankful to Liang Shi, Heng Zhong and Kasi Azhakanandam for project support and technical discussions, respectively.
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The study was funded by Syngenta’s internal resources.
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Sivamani, E., Nalapalli, S., Prairie, A. et al. A study on optimization of pat gene expression cassette for maize transformation. Mol Biol Rep 46, 3009–3017 (2019). https://doi.org/10.1007/s11033-019-04737-3
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DOI: https://doi.org/10.1007/s11033-019-04737-3