Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins.

摘要: Parasporal crystalline inclusions of Bacillus thuringiensis subspecies are among the most promising bacterial biopesticides available for use today. As a whole, these proteins (B. toxins) demonstrate great specificity toward certain orders insects and, to date, have shown no known side effects nontarget animals. Currently, one major drawbacks B. toxins as externally applied is their lack persistence in field due factors such rain washout and degradation by UV irradiation. In part, problems being addressed production transgenic food textile crops expressing toxin genes own tissues. Agricultural biotechnology companies pursuing methodologies hope producing which will be resistant insect pests without need pesticides. To trials plants resulted mixed success due, attractive advantages pesticides, i.e., narrow spectrum toxicity. This situation can engineering more than or novel with multiple specificities. increases, however, resistance may become problem. A few species already demonstrated increased tolerance toxins, either laboratory. suggested studies, decreased susceptibility variety factors, including alterations gut physiology (14, 30, 37) ligand binding characteristics receptor(s) involved (8, 32, 47). To understand development receptor-mediated resistance, investigators first identify characterize physiologically important receptor molecules each class from background low-affinity proteins. That specific protein receptors Cry killing target has been since mid-1980s. Studies radiolabeled suspensions midgut isolated various procedures generated rather extensive list putative (4, 7, 15, 26, 41, 45, 46) simultaneously identifying protein(s) question sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) blots. SDS-PAGE blots, when incubated at least permit visual estimation both number molecular masses. Ligand blots Manduca sexta used successfully partially this particular Cry1A lepidopteran-specific (3, 10, 11, 19, 23, 24, 33, 42, 43). Cry1Aa, Cry1Ab, Cry1Ac 82 90% amino acid identity another compared directly, exhibit indistinguishable toxicities M. larvae (16, 17, 45). Cry1Aa Cry1Ab recognize single sexta, 210-kDa cadherin-like glycoprotein BT-R1 (42, 43). For Cry1Ac, two populations identified blot analyses whole preparations, (10, 28, 33) other masses ranging 85 120 kDa (5, 21, 44). aminopeptidases approximately now subjected partial purification, described some detail 12, 13, 29, 34, 39). Both 120-kDa aminopeptidase also cloned, complementary DNA sequences reported (22, 43). BT-R1 specifically bind high affinity three tested (Cry1Aa, Cry1Ac) preparations (19, heterologous cell cultures cDNA (19). only that Cry1A-specific expressed mammalian cultures. binds Cry1Aa, extremely virtually equal affinities specificities homologous competition experiments membrane prepared larval midguts transiently transfected Sf21 cells The report were show positive correlation between immobilized on polyvinylidene difluoride (PVDF) filters. paramount importance understanding conflicting reports appear data indirectly identification given pointed out recently Lee Dean (27). To resolve confusion surrounding relevant thuringiensis, we designed different protocols determine whether affect results our work clearly common high-affinity properties not affected any commonly accepted study ligand-receptor lepidopteran insects.