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Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16

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Abstract

To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5–6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

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Acknowledgments

We thank Sheryl Hubbard for her technical assistance and are grateful to the financial support from the Ontario Soybean Board and Eastern Cereal and Oilseed Research Centre. Agriculture and Agri-Food Canada Contribution # 13-468.

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  1. Yunfang Zhang

    Present address: Shanghai Institute of Biological Science, China Academic Science, 320 Yueyang Road, Life Science Building, Shanghai, 200032, China

Authors and Affiliations

  1. Eastern Cereals and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, ON, K1A0C6, Canada

    Yunfang Zhang, Johann Schernthaner, Natalie Labbé & Daina H. Simmonds

  2. Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, ON, K1A 0L2, Canada

    Mary A. Hefford

  3. Ball Helix, Ball Horticultural Company, 622 Town Road, West Chicago, IL, 60185-2698, USA

    Jiping Zhao

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  1. Yunfang Zhang
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Zhang, Y., Schernthaner, J., Labbé, N. et al. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16. Transgenic Res 23, 455–467 (2014). https://doi.org/10.1007/s11248-013-9777-5

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