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Evaluating the effect of codon optimization on expression of bar gene in transgenic tobacco plants

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Abstract

Information of codon usage bias has been used for modifying genes for improved expression in heterologous systems. Codon modifications are carried out using the species-specific codon usage so that they reflect the codon usage pattern of the host, without modifying the amino acid sequence of the encoded protein. In the present study we analyzed the effect of codon optimization on the expression of the bacterial gene bar in tobacco. In order to identify the percentage of optimal codons needed to achieve high levels of protein expression, bar genes with different percentage and placement of optimal codons were analyzed in transgenic tobacco lines. It was observed that there was no gain in bar protein expression when the percentage of optimal codons was increased from 63.9% (as observed in the wild type bar gene) to 93.9%. However, reducing the percentage of optimal codons to 54.0 led to a drop in the levels of the bar protein. Further, in silico analysis was also carried out in ~ 4500 genes present on chromosome 2 of Arabidopsis thaliana to study the distribution of optimal codons. It was observed that majority (88%) of the genes have 30–50% of optimal codons and none of the gene was found to have more than 80% of optimal codons. The present study showed that there may not be a one-to-one correlation between the percentage of optimal codons and the expression levels of the transgene. A certain percentage of optimal codons is probably enough to achieve high levels of transgene expression. Any increase in the percentage of optimal codons beyond this level may not necessarily lead to any further improvement in expression.

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Abbreviations

CUB:

Codon usage bias

PAT:

Phosphinothricin acetyl transferase

PPT:

Phosphinothricin

RSCU:

Relative synonymous codon usage

URM:

Upstream regulatory module

CUTG:

Codon usage tabulated from GenBank

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Acknowledgement

The work was supported by grant in aids from University Grants Commission, India under their Special Assistance Program and from the University of Delhi. PA was supported by a fellowship from Council for Scientific and Industrial Research.

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  1. Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi, 110021, India

    Parul Agarwal, Taru Gautam, Amarjeet Kumar Singh & Pradeep Kumar Burma

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  1. Parul Agarwal
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  2. Taru Gautam
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  3. Amarjeet Kumar Singh
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  4. Pradeep Kumar Burma
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PA and PKB conceived and designed the experiments, analyzed data and wrote the manuscript. PA carried out the experiments. TG and AKS contributed to plant transformation and Southern hybridization, respectively.

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Correspondence to Pradeep Kumar Burma.

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Agarwal, P., Gautam, T., Singh, A.K. et al. Evaluating the effect of codon optimization on expression of bar gene in transgenic tobacco plants. J. Plant Biochem. Biotechnol. 28, 189–202 (2019). https://doi.org/10.1007/s13562-019-00506-2

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