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Two variants of the promoter of the squash aspartic acid protease inhibitor multigene family were isolated from Cucurbita maxima cv. ‘Supermarket’ Hybrid genomic DNA. The isolated promoters, possibly not full length, comprised a 5′-untranslated region (UTR) of 202–208 bp, contained a 63-bp upstream open reading frame (uORF) and the immediate upstream sequences of 441–445 bp. The two promoters contained several small deletions relative to each other and 22 single base differences but exhibit overall 92.5% homology over 654 bp. When the promoters were fused to a β-glucuronidase reporter gene and expressed in tobacco, one variant was highly expressed in the companion cells of the inner and outer phloem of leaves and at lower levels in other organs. The other variant was expressed at high levels in the long glandular trichomes of the leaf. Deletion analysis identified a region of ~280 bp immediately upstream of the 5′-UTR containing the TATA box that was responsible for phloem specific expression and a further region of ~180 bp that enhanced expression in one promoter and conferred trichome expression in the other. Removal of the 5′-UTR, including the uORF, inactivated the phloem promoter.

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.

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.

The effects of concanavalin A (ConA), a glucose/mannose-specific lectin from jackbean (Canavalia ensiformis), on insect crop pests from two different orders, Lepidoptera and Homoptera, were investigated. When fed to larvae of tomato moth (Lacanobia oleracea) at a range of concentrations (0.02–2.0% of total protein) in artificial diet, ConA decreased survival, with up to 90% mortality observed at the highest dose level, and retarded development, but had only a small effect on larval weight. When fed to peach-potato aphids (Myzus persicae) at a range of concentrations (1–9μM) in liquid artificial diet, ConA reduced aphid size by up to 30%, retarded development to maturity, and reduced fecundity (production of offspring) by >35%, but had little effect on survival. With both insects, there was a poor correlation between lectin dose and the quantitative effect. Constitutive expression of ConA in transgenic potatoes driven by the CaMV 35S promoter resulted in the protein accumulating to levels lower than predicted, possibly due to potato not being able to adequately reproduce the post-translational processing of this lectin which occurs in jackbean. However, the expressed lectin was functionally active as a haemagglutinin. Bioassay of L. oleracea larvae on ConA-expressing potato plants showed that the lectin retarded larval development, and decreased larval weights by >45%, but had no significant effect on survival. It also decreased consumption of plant tissue by the larvae. In agreement with the diet bioassay results, ConA-expressing potatoes decreased the fecundity of M. persicae by up to 45%. ConA thus has potential as a protective agent against insect pests in transgenic crops.

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

A cDNA library was prepared from, poly(A)+ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10-5% of poly(A)+ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)+RNA population.