Characterisation of midgut digestive proteases from the maize stem borer Busseola fusca

Authors

  • Derick George,

    1. School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK
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  • Natalie Ferry,

    1. School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK
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  • Emma J Back,

    1. School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK
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  • Angharad MR Gatehouse

    Corresponding author
    1. School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK
    • School of Biological Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
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Abstract

BACKGROUND: Insect damage is a major constraint on maize production. Control of Busseola fusca (Fuller) in sub-Saharan Africa is relatively ineffective; the major larval digestive enzymes were characterised with a view to developing future control strategies.

RESULTS: Using BODIPY-FL Casein, maximal activity was at pH 9.5, with six protease forms visualised by gelatin-PAGE. Synthetic substrates and diagnostic inhibitors demonstrated the presence of serine proteases. Chymostatin was a potent inhibitor of general proteolysis (90%), providing strong evidence for the presence of chymotrypsin; it also caused significant inhibition (>95%) with SA2PFpNA as substrate. The I50 values for chymostatin with casein and SA2PFpNA were 0.0075 µM and 0.06 µM respectively. Z-Phe-Arg-pNA activity was inhibited by chymostatin and TLCK (50 and 30% respectively), suggesting the presence of trypsin-like activity. BApNA hydrolysis was also strongly inhibited by chymostatin and TLCK (92 and 75%), suggesting trypsin activity, while SBBI, PMSF, pepstatin and E-64 had no significant effect. Interestingly, SBBI (I50 = 0.39 µM) and SBTI both inhibited general proteolysis by approximately 70%, suggesting that SBBI's dual inhibitory role makes this inhibitor a potentially useful candidate for expression in maize for control of B. fusca.

CONCLUSION: These results provide a basis for the rational design of insect-resistant transgenic maize expressing protease inhibitors. Copyright © 2008 Society of Chemical Industry

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