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Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed

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Abstract

A mutant Bowman-Birk gene was created that encoded an inactive high-sulfur product. It was used to transform soybean line Asgrow 3237. Transformants bearing the mutant gene were identified by GUS expression, PCR analysis, and Southern analysis. The amount of steady state mRNA from the mutant gene in the transformed plants showed that the gene was highly expressed, but the amount of message from the unmodified Bowman-Birk gene did not change detectably. Proteins synthesized at the direction of the mutant Bowman-Birk gene accumulated in seeds of the transformed plants, and there was a marked decrease in the ability of extracts prepared from these seeds to inhibit trypsin and chymotrypsin despite the presence of Kunitz trypsin inhibitor. The more prevalent mRNA from the mutant gene was considered to out-compete message from the native genes to decrease the amount of active Bowman-Birk inhibitor.

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Abbreviations

BBI:

Bowman-Birk inhibitor

KTI:

Kunitz inhibitor

DAF:

Days after flowering

IEF:

Isoelectric focusing

Q-TOF:

Quadruple time-of-flight mass spectrometry

BAPNA:

N-α-Benzoyl-dl-Arginine p-Nitroanilide

GPNA:

N-Glutaryl-L-Phenylalanine p-Nitroanilide

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Acknowledgments

We would like to thank Dr. Leslie Hicks of the Danforth Center for her expertise with the Q-TOF MS/MS analysis. Financial support from the American Soybean Association, the North Central Soybean Research Program, and the United Soybean Board at various times during these experiments is gratefully acknowledged. Mention of a product used during the description of these experiments does not constitute an endorsement of this product to the exclusion of other products by USDA-ARS.

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Correspondence to Niels C. Nielsen.

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Livingstone, D., Beilinson, V., Kalyaeva, M. et al. Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed. Plant Mol Biol 64, 397–408 (2007). https://doi.org/10.1007/s11103-007-9163-x

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  • DOI: https://doi.org/10.1007/s11103-007-9163-x

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