title: The expression of a mammalian proteinase inhibitor, bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae.
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.
authors.affiliation: The Horticulture and Food Research Institute of New Zealand, Palmerston North Research Centre. jchristeller@hort.cri.nz
authors.forename: John T
authors.initials: JT
authors.lastname: Christeller
authors.forename: Elisabeth P J
authors.initials: EP
authors.lastname: Burgess
authors.forename: Valentina
authors.initials: V
authors.lastname: Mett
authors.forename: Heather S
authors.initials: HS
authors.lastname: Gatehouse
authors.forename: Ngaire P
authors.initials: NP
authors.lastname: Markwick
authors.forename: Colleen
authors.initials: C
authors.lastname: Murray
authors.forename: Louise A
authors.initials: LA
authors.lastname: Malone
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Wright
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Philip
authors.forename: Dianne
authors.initials: D
authors.lastname: Watt
authors.forename: Laurence N
authors.initials: LN
authors.lastname: Gatehouse
authors.forename: Gábor L
authors.initials: GL
authors.lastname: Lövei
authors.forename: April L
authors.initials: AL
authors.lastname: Shannon
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Phung
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Watson
authors.forename: William A
authors.initials: WA
authors.lastname: Laing
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D002417:Cattle; D005822:Genetic Vectors; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects*; D010572:Pest Control, Biological; D030821:Plants, Genetically Modified; D013154:Spleen / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics* / Q000469:parasitology; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism* / Q000633:toxicity
chemical_list: D014361:Trypsin Inhibitors; D014357:Trypsin