Abstract
The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3′ and 5′ changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
An G, Ebert PR, Mitra A and Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoot RA and Verna DPS (eds), Plant Molecular Biology Manual. Vol. A3 (pp. 1–199) Kluwer Academic Publishers, Dordrecht.
Anderson S and Kingston IB (1983) Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe. Proc Natl Acad Sci USA 80: 6838–6842.
Bekkaoui F, Pilon N, Laine E, Raju DSS, Crosby WL and Dunstan DI (1988) Transient gene expression in electroporated Picea glauca protoplasts. Plant Cell Rep 7: 481–484.
Bevan, M (1984) Binary agrobacterium vectors for plant transformation. Nucl Acids Res 22: 8711–8721.
Bown DP, Wilkinson HS and Gatehouse JA (1997) Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 27: 625–638.
Burgess EPJ and Gatehouse AMR (1997) Engineering for insect pest resistance. In: McKersie BD and Brown DCW (eds), Biotechnology and the Improvement of Forage Legumes. (pp. 229–258) Biotechnology in Agriculture Series, No. 17, CAB International.
Burgess EPJ, Main CA, Stevens PS, Gatehouse AMR, Christeller JT and Laing WA (1993) Protease inhibitors active against porina caterpillar (Wiseana cervinata). In: Prestidge RA (ed.), Proc 6th Australasian Grassl Invert Ecol Conf AgResearch. (pp. 331–339) Hamilton, New Zealand.
Burgess EPJ, Main CA, Stevens PS, Christeller JT, Gatehouse AMR and Laing WA (1994) Effects of protease inhibitor concentration and combinations on the survival, growth and gut enzyme activities of the black field cricket, Teleogryllus commodus. J Insect Physiol 40: 803–811.
Cavener DR and Ray SC (1991) Eukaryotic start and stop translation sites. Nucl Acids Res 19: 3185–3192.
Christeller JT, Laing WA, Markwick NP and Burgess EPJ (1992) Midgut protease activities in 12 phytophagous lepidopteran larvae: dietary and protease inhibitor interactions. Insect Biochem Molec Biol 22: 735–746.
Christeller JT and Shaw BD (1989) The interaction of a range of serine protease inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 19: 233–241.
Creighton TE and Charles IG (1987) Sequences of the genes and polypeptide precursors for two bovine protease inhibitors. J Mol Biol 194: 11–22.
Datla RSS, Bekkaovic F, Hammelindl JK, Pilate G, Dunstan DI and Crosby WL (1993) Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci 94: 130–149.
Ditta G, Stanfield S, Corbin D and Helinski DR (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351.
Fan X, Shi X, Zhao J, Zhao R and Fan Y (1999) Insecticidal activity of transgenic tobacco plants expressing both Bt and CpTI genes on cotton bollworm (Helicoverpa armigera). Chin J Biotechnol 15: 1–5.
Fioretti E, Binotti I, Barra D, Citro G, Ascoli F and Antonini E (1983) Heterogeniety of the basic pancreatic trypsin inhibitor (Kunitz) in various bovine organs. Eur J Biochem 130: 13–18.
Gatehouse LN, Shannon A, Burgess EPJ and Christeller JT (1997) Characterization of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to 4 proteinase inhibitors in the diet. Insect Biochem Mol Biol 27: 929–944.
Gebhard W, Tschesche H and Fritz H (1986) Biochemistry of aprotinin and aprotinin-like inhibitors. In: Barrett AJ and Salvesen G (eds), Proteinase Inhibitors. (pp. 375–388) Elsevier Science Publishers, Amsterdam.
Girard C, Le Métayer M, Bonadé-Bottino M, Pham-Delègue M-H and Jouanin L (1998) High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Molec Biol 28: 229–237.
Giri AP, Harsulkar AM, Deshpande VV, Sainani MN, Gupta VS and Ranjekar PK (1988) Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 116: 393–401.
Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molec Biol 20: 1203–1207.
Goldenberg DP (1988) Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and-38 thiols. Biochem 27: 2481–2489.
Heath RL, McDonald G, Christeller JT, Lee M, Bateman K, West J et al. (1997) Protease inhibitors from Nicotiana alata enhance plant resistance to insect pests. J Insect Physiol 43: 833–842.
Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF and Boulter D (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 30: 160–163.
Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG and Fraley RF (1985) A simple and general method for transferring genes into plants. Science 227: 1299–1301.
Johnston KA, Gatehouse JA and Anstee JH (1993) Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera. J Insect Physiol 39: 657–664.
Johnston KA, Lee MJ, Brough C, Hilder VA, Gatehouse AMR and Gatehouse JA (1995) Protease activities in the larval midgut of Heliothis virescens: evidence for trypsin and chymotrypsin-like enzymes. Insect Biochem Molec Biol 25: 375–383.
Jongsma MA, Bakker PL, Peters J, Bosch D and Stiekema WJ (1995) Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 92: 8041–8045.
Joshi CP (1987) An inspection of the domain between the putative TATA box and the translation start site in 79 plant genes. Nucl Acids Res 15: 6643–6653.
Kassell B and Laskowski M (1965) The basic inhibitor of bovine pancreas. V. The disulphide linkages. Biochem Biophys Res Commun 20: 463–468.
Kassell B and Wang T-W (1971) The action of thermolysin on the basic trypsin inhibitor of bovine organs. In: Fritz H and Tschesche H (eds), Proc Int Res Conf Proteinase Inhibitors. (pp. 89–94) Munich, November 1970, W.de Gruyter, Berlin.
Kosak M (1996) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cellulose 44: 283–292.
Kosiel MG, Carozzi NB and Desai N (1986) Optimizing expression of transgenes with an emphasis on post-transcriptional events. Plant Mol Biol 32: 393–405.
Kraut H, Frey EK and Werle E (1930) Uber die Inaktivierung des Kallikreins. Hoppe-Seyler's Z Physiol Chem 192: 1–21.
Kunitz M and Northrop JH (1936) Isolation from beef pancreas of crystalline trypsinogen, trypsin, trypsin inihibitor and an inhibitor trypsin compound. J Gen Physiol 19: 991–1007.
Kurland CG (1991) Codon bias and gene expression. FEBS Lett 285: 165–169.
Markwick NP, Laing WA, Christeller JT, McHenry JZ and Newton MR (1998) Overproduction of digestive enzymes compensates for inhibitory effects of protease and α-amylase inhibitors fed to three species of leafrollers (Lepidoptera: Tortricidae). J Econ Entomol 91: 1265–1276.
May RM (1993) Resisting resistance. Nature 361: 593–594.
McGaughey WH and Whalon ME (1992) Managing insect res-istance to Bacillus thuringiensis toxins. Science 258: 1451–1455.
McManus MT and Burgess EPJ (1995) Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 41: 731–738.
Murray EE, Lotzer J and Eberle M (1989) Codon usuage in plant genes. Nucl Acids Res 17: 477–798.
Payne RW, Lane PW, Digby PGN, Harding SA, Leech PK, Morgan GW et al. (1993) Genstat 5 Release 3 Reference Manual. Lawes Agricultural Trust, Oxford: Clarendon Press.
Sanchez-Serrano J, Schmidt R, Schell J and Willmitzer L (1986) Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol Gen Genet 203: 15–20.
Uriel J and Berges J (1968) Characterization of natural inhibitors of trypsin and chymotrypsin by electrophoresis in acrylamide-agarose gels. Nature 218: 578–580.
Walker-Simmons M and Ryan CA (1977) Immunological identification of proteinase inhibitors I and II in isolated tomato leaf vacuoles. Plant Physiol 60: 61–63.
Wandelt CE, Khan MRI, Craig S, Schroeder HE, Spencer D and Higgins TJV (1992) Vicillin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in leaves of transgenic plants. Plant J 2: 181–192.
Weissman JS and Kim PS (1992) The pro region of BPTI facilitates folding. Cell 71: 841–851.
Wu Y, Llewellyn D, Mathews A and Dennis E (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molec Breed 3: 371–380.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Christeller, J.T., Burgess, E.P., Mett, V. et al. The Expression of a Mammalian Proteinase Inhibitor, Bovine Spleen Trypsin Inhibitor in Tobacco and its Effects on Helicoverpa armigera Larvae. Transgenic Res 11, 161–173 (2002). https://doi.org/10.1023/A:1015210919077
Issue Date:
DOI: https://doi.org/10.1023/A:1015210919077