Hostname: page-component-8448b6f56d-c4f8m Total loading time: 0 Render date: 2024-04-25T06:09:10.995Z Has data issue: false hasContentIssue false
  • English
  • Français

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Published online by Cambridge University Press:  22 October 2009

Hang Thu Nguyen  and
Johannes A. Jehle
Hang Thu Nguyen
Affiliation:
Agricultural Service Center Palatinate (DLR Rheinpfalz), Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany
Johannes A. Jehle
Affiliation:
Agricultural Service Center Palatinate (DLR Rheinpfalz), Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 °C, –20 °C, and –80 °C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 °C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at –20 °C and –80 °C was observed. Bioassays with neonate larvae of Ostrinianubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 °C for four months showed a significantly decreasing activity measured as median lethal concentration (LC50), whereas the protein activity declined less than 11-fold after two years storage at –20 °C. When stored at –80 °C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC50 values of 7–10 ng Cry1Ab per cm2 diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at –80 °C is recommended.

Keywords

Bt cornOstrinia nubilalisresistance monitoringbioassay
Type
Research Article
Information
Environmental Biosafety Research , Volume 8 , Issue 2 , April 2009 , pp. 113 - 119
Copyright
© ISBR, EDP Sciences, 2009

References

Abbott, WS (1925) A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 266–267 CrossRef
Bosch D, Visser B, Stiekema WJ (1994) Analysis of non-active engineered Bacillus thuringiensis crystal proteins. FEMS Microbiol. Lett. 118: 129–134
Bradford, M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72: 248–254 CrossRef
Brookes G, Barfoot P (2008) GM crops: global socio-economic and environmental impacts 1996–2006. PG Economics Ltd, UK, 20 p
Chaufaux, J, Segui, M, Swanson, JJ, Bourguet, D, Siegfried, BD (2001) Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis protein. J. Econ. Entomol. 94: 1564–1570 CrossRef
Crespo, ALB, Spencer, AT, Nelk, E, Pusztai-Carey, M, Moar, JW, Siegfried, BD (2008) Comparison and validation of methods to quantify Cry1Ab protein from Bacillus thuringiensis for standardization of insect bioassays. Appl. Environ. Microbiol. 74: 130–135 CrossRef
De Maagd RA, Kwa MSG, Van der Klei H, Yamamoto T, Schipper B, Vlak JM, Stiekema WJ, Bosch D (1996) Domain III substitution in Bacillus thuringiensis Delta-Endotoxin Cry1A(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. Appl. Environ. Microbiol. 65: 1537–1543
EPA (1998) The Environmental Protection Agency's White Paper on Bt Plant-pesticide resistance management. US Environmental Protection Agency, Washington, DC, USA
Farinós, GP, de la Poza, M, Hernández-Crespo, P, Ortego, F, Castañera, P (2004) Resistance monitoring of field populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain. Entomol. Exp. Appl. 110: 23–30 CrossRef
Gonzáles-Núñez, M, Otergo, F, Castañera, P (2000) Susceptibility of Spanish populations of the corn borers Sesamia nonagrioides (Lepidoptara: Noctuidae) and Ostrinia nubilalis (Lepidoptera: Crambidae) to a Bacillus thuringiensis endoprotein. J. Econ. Entomol. 93: 459–463 CrossRef
Gould, F (1998) Sustainability of transgenic insecticidal cultivars: Intergrating pest genetics and ecology. Annu. Rev. Entomol. 43: 701–726 CrossRef
Höfte, H, de Greve, H, Seurinck, J, Jansens, S, Mahillon, J, Ampe, C, Vandekerckhove, J, Vanderbruggen, H, van Montagu, M, Zabeau, M, Vaek, M (1986) Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161: 273–280 CrossRef
Höss, S, Arndt, M, Baumgarte, S, Tebbe, CC, Nguyen, TH, Jehle, JA (2007) Effect of transgenic corn and Cry1Ab protein on the nematode, Caenorhabditis elegans. Ecotox. Environ. Saf. 70: 334–340 CrossRef
Huang, F, Higgins, RA, Buschman, LL (1997) Baseline susceptibility to Bacillus thuringiensis subsp. kurstaki under selection pressure in European corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 90: 1137–1143 CrossRef
Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 CrossRef
Marçon, PCRG, Young, LG, Steffey, KL, Siegfried, BD (1999) Baseline susceptibility of European corn borer (Lepidoptera: Crambidae) to Bacillus thuringiensis proteins. J. Econ. Entomol. 92: 279–285 CrossRef
Marçon, PCRG, Siegfried, BD, Spencer, T, Hutchison, WD (2000) Development of diagnostic concentrations for monitoring Bacillus thuringiensis resistance in European corn borer (Lepidoptera: Crambidae). J. Econ. Entomol. 93: 925–930 CrossRef
Martens, JWM, Visser, B, Vlak, JM, Bosch, D (1995) Mapping and characterisation of the entomocidal domain of the Bacillus thuringiensis Cry1Ab protoxin. Mol. Gen. Genet. 247: 482–487 CrossRef
Meise, T, Langenbruch, GA (2007) Susceptibility of German populations of the Corn Borer Ostrinia nubilalis (Lepidoptera: Pyralidae) to a Bacillus thuringiensis endoprotein. Nachrichtenbl. Deut. Pflanzenschutzd. 59: 297–301
Nguyen, TH, Meise, T, Langenbruch, GA, Jehle, JA (2004) Production of Cry1Ab toxin in E. coli for standardisation of insect bioassays. WRPS/IOBC Bulletin 27: 125–130
Roush, RT, Miller, GL (1986) Considerations for design of insecticide resistance monitoring programs. J. Econ. Entomol. 79: 293–298 CrossRef
Saeglitz, C, Bartsch, D, Eber, S, Gathmann, A, Priesnitz, KU, Schuphan, I (2006) Monitoring the Cry1Ab susceptibility of European corn borer in Germany. J. Econ. Entomol. 99: 1768–1773 CrossRef
Siegfried, BD, Marçon, PCRG, Witkowski, JF, Wright, RJ, Warren, GW (1995) Susceptibility of field populations of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae), to the microbial insecticide Bacillus thuringiensis Berliner. J. Agri. Entomol. 12: 257–263
Smith RW (1997) Visual hypothesis testing with confidence intervals. In Proceedings of the Twenty-Second Annual SAS® Users Group International Conference, Cary NC ed, SAS Institute, pp 1252–1257. http://www2.sas.com/proceedings/sugi22/STATS/PAPER270.PDF
Tabashnik, BE, Gassmann, AJ, Crowder, DW, Carrière, Y (2008) Insect resistance to Bt crops: evidence versus theory. Nat. Biotech. 26: 199–202 CrossRef
Unkelbach HD, Wolf T (1985) Qualitative Dosis-Wirkungsanalyse. Gustav Fischer Verlag Stuttgart, p 45, 106