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Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

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Abstract

The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5 kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4–7) in comparison with Tia (pI 4–5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.

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Abbreviations

2-DE:

Two-dimensional polyacrylamide gel electrophoresis

2-DN:

Two-dimensional native polyacrylamide gel electrophoresis

2-DZ:

Two-dimensional zymography

CBB:

Coomassie brilliant blue R-250

IEF:

Isoelectric focusing

IPG:

Immobilized pH gradient

pI:

Isoelectric point

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgments

This research was supported by the Technology Development Program for Agriculture and Forestry, the Ministry for Food, Agriculture, Forestry, and Fisheries, the Korea Atomic Energy Research Institute (KAERI) and the Ministry of Education, Science and Technology (MEST), Korea.

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Authors and Affiliations

  1. Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong, Jeongeup, 580-185, Republic of Korea

    K. J. Lee, J.-B. Kim, B.-K. Ha, S. H. Kim, S.-Y. Kang & D. S. Kim

  2. Department of Plant Biotechnology, Dongguk University, Seoul, Republic of Korea

    K. J. Lee & B.-M. Lee

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  1. K. J. Lee
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  2. J.-B. Kim
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  3. B.-K. Ha
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  4. S. H. Kim
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  5. S.-Y. Kang
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  6. B.-M. Lee
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  7. D. S. Kim
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Correspondence to B.-M. Lee or D. S. Kim.

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Lee, K.J., Kim, JB., Ha, BK. et al. Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]. Amino Acids 43, 379–388 (2012). https://doi.org/10.1007/s00726-011-1092-y

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  • DOI: https://doi.org/10.1007/s00726-011-1092-y

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