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Overexpression of d-amino acid oxidase from Bradyrhizobium japonicum, enhances resistance to glyphosate in Arabidopsis thaliana

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Abstract

Key message

The glyphosate resistance in Escherichia coli and Arabidopsis was due to d -amino acid oxidase expression.

Abstract

Transgenic glyphosate-resistant crops have a high percentage in the total area devoted to transgenic crops worldwide. d-amino acid oxidase (DAAO) can metabolize glyphosate by oxidative cleavage of the carbon–nitrogen bond on the carboxyl side and yield aminomethyl phosphonic acid and glyoxylate, which are less toxic to plants than glyphosate. To date, reports on the use of DAAO to enhance glyphosate resistance in plants are lacking. In this paper, we report synthesis, and codon usage optimization for plant expression, of the DAAO gene by successive polymerase chain reaction from Bradyrhizobium japonicum. To confirm the glyphosate resistance of the DAAO gene, the recombinant plasmid pYPX251 (GenBank Accession No: AY178046) harboring the wild-type DAAO gene was transformed into DH5α. The positive transformants grew well both on solid and in liquid M9 medium containing 200 mM glyphosate. The optimized DAAO gene was transformed into Arabidopsis and 9 days after application of 10 mM glyphosate, the 4-week-old wild-type plants all died; by contrast, transgenic plants could grow normally. The proline content and peroxidase activity showed that glyphosate could induce proline accumulation and produce reactive oxygen species.

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Abbreviations

AMPA:

Aminomethyl phosphonic acid

DAAO:

d-amino acid oxidase

EPSPS:

5-Enolpyruvylshikimate-3-phosphate synthase

GAT:

Glyphosate acetyl-transferase

GO:

Glycine oxidase

GOX:

Glyphosate oxidoreductase

PCR:

Polymerase chain reaction

POD:

Peroxidase

p251:

pYPX251

ROS:

Reactive oxygen species

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Acknowledgments

The research was supported by the International Scientific and Technological Cooperation (13440701700), Agriculture Science Technology Achievement Transformation Fund (133919N1300), Young Foundation of Shanghai Academy of Agricultural Science (2010-14), National Natural Science Foundation (31200075, 31200076, 31200212). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai, 201106, People’s Republic of China

    Hongjuan Han, Bo Zhu, Xiaoyan Fu, Shuanghong You, Bo Wang, Zhenjun Li, Wei Zhao, Rihe Peng & Quanhong Yao

  2. College of Horticulture Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

    Shuanghong You & Quanhong Yao

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  1. Hongjuan Han
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  2. Bo Zhu
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Correspondence to Quanhong Yao.

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We declare that we have no conflict of interest.

Additional information

Communicated by L. Peña.

Electronic supplementary material

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299_2015_1850_MOESM1_ESM.docx

Supplemental Table 1 Sequences for all primer sets used for chemical synthesis of DAAO gene according to plant codon usage bias. Letters in boxes are restriction endonuclease sites (DOCX 12 kb)

Fig. S1 The alignment result of the optimal sequence to the wild-type DAAO gene given by a BLAST search (PDF 129 kb)

299_2015_1850_MOESM3_ESM.jpg

Fig. S2 RT-PCR analysis of the expression of DAAO (target gene) in other transgenic lines (Do1-15 except for Do3, Do6 and Do14) (JPEG 43 kb)

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Han, H., Zhu, B., Fu, X. et al. Overexpression of d-amino acid oxidase from Bradyrhizobium japonicum, enhances resistance to glyphosate in Arabidopsis thaliana . Plant Cell Rep 34, 2043–2051 (2015). https://doi.org/10.1007/s00299-015-1850-5

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