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
References
Bernard RL, Hymowitz T (1986) Registration of L81-4590, L81-4871, and L83-4387 soybean germplasm lines lacking the Kunitz trypsin inhibitor. Crop Sci 26:650–651
Biermann BJ, de Banzie JS, Handelsman J, Thompson JF, Madison JT (1998) Methionine and sulfate increase a Bowman-Birk type protease inhibitor and its messenger RNA in soybeans. J Agric Food Chem 46:2858–2862
Birk Y (1985) The Bowman-Birk inhibitor. Int J Pept Protein Res 25:113–131
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Clarke EJ, Wiseman J (2000) Developments in plant breeding for improved nutritional quality of soya beans II. Anti-nutritional factors. J Ag Sci 134:125–136
Cook DA, Jensen AH, Farley JR, Hymowitz T (1988) Utilization by growing and finishing pigs of raw soybeans of low Kunitz trypsin inhibitor content. J Anim Sci 66:1686–1691
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Phys 129:13–22
Gillespie JM, Blagrove RJ, Randall PJ (1978) Effect of sulfur supply on the seed globulin composition of various species of lupin. Aust J Plant Physiol 5:641–650
Hajduch M, Ganapathy A, Stein JW, Thelen JJ (2005) A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database. Plant Phys 137:1397–1419
Higgins TJ, Chandler PM, Randall PJ, Spencer D, Beach LR, Blagrove RJ, Kortt AA, Inglis AS (1986) Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds. J Bio Chem 261:11124–11130
Hinchee M, Connor-Ward D, Newell C, McDonnell R, Sato S, Grasser C, Fischhoff D, Re D, Fraley R, Horsch R (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. BioTechnology 6:915–922
Holowach LP, Madison JT, Thompson JF (1986) Studies on the mechanism of regulation of the mRNA level for a soybean storage protein subunit by exogenous L-methionine. Plant Phys 80:561–567
Holowach LP, Thompson JF, Madison JT (1984) Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro. Plant Phys 74:576–583
Hurkman WJ, Tanaka CK (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Phys 81:802–806
Hwang DL-R, Lin K-TD, Yang W-K, Foard DE (1977) Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean. Biochim Biophys Acta 495:369–382
Jofuku KD, Goldberg RB (1989) Kunitz Trypsin inhibitor genes are differentially expressed during the Soybean life cycle and in transformed tobacco plants. Plant Cell 1:1079–1093
Jung R, Scott MP, Oliveira LO, Nielsen NC (1992) A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA. Gene 121:17–24
Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302
Kowalski D, Laskowski M Jr (1976) Chemical-enzymatic replacement of Ile64 in the reactive site of soybean trypsin inhibitor (Kunitz). Biochemistry 15:1300–1309
Laskowski M Jr (1986) Protein inhibitors of serine proteinases-mechanism and classification. Adv Exp Med Biol 199:1–17
Laskowski M Jr Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626
Molvig L, Tabe LM, Eggum BO, Moore AE, Craig S, Spencer D, Higgins TJV (1997) Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. PNAS 94:8393–8398
Narasimhulu SB, Deng X-b, Sarria R, Gelvin SB (1996) Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8:873–886
Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812
Olhoft P, Flagel L, Donovan C, Somers D (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735
Orf JH, Hymowitz T (1979) Genetics of the Kunitz trypsin inhibitor: an antinutritional factor in soybeans. J Am Oil Chem Soc 56:722–726
Perez-Maldonado RA, Mannion PF, Farrell DJ (2003) Effects of heat treatment on the nutritional value of raw soybean selected for low trypsin inhibitor activity. Br Poult Sci 44:299–308
Sambrook J, Russell DW (2001) Molecular cloning-a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sealock RW, Laskowski M Jr (1969) Enzymatic replacement of the arginyl by a lysyl residue in the reactive site of soybean trypsin inhibitor. Biochemistry 8:3703–3710
Schroeder HE (1982) Quantitative studies on the cotyledonary proteins in the genus Pisum. J Sci Food Agric 33:623–633
Sheffield J, Taylor N, Fauquet C, Chen S (2006) The Cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression. Proteomics 6:1588–1598
Slightom JL, Sun SM, Hall TC (1983) Complete nucleotide sequence of a French bean storage protein gene: phaseolin. PNAS 80:1897–1901
Spencer RW, Foard DE, Larkins BA (1984) Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean. J Biol Chem 259:9883–9890
Streit LG, Beach LR, Register JC III, Jung R, Fehr WR (2001) Association of the Brazil nut protein gene and Kunitz trypsin inhibitor alleles with soybean protease inhibitor activity and agronomic traits. Crop Sci 41:1757–1760
Tan-Wilson AL, Chen JC, Duggan MC, Chapman C, Obach RS, Wilson KA (1987) Soybean Bowman-Birk trypsin isoinhibitors: classification and report of a glycine-rich trypsin inhibitor class. J Agric Food Chem 35:974–981
Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO 6:2519–2523
Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250
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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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