Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes fr
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ORIGINAL PAPER
Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis Vadim Khasdan · Maria Sapojnik · Arieh Zaritsky · A. Rami Horowitz · Sammy Boussiba · Mario Rippa · Robert Manasherob · Eitan Ben-Dov
Received: 8 April 2007 / Revised: 19 June 2007 / Accepted: 11 July 2007 / Published online: 31 July 2007 © Springer-Verlag 2007
Abstract The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding -endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine
Communicated by Jan Roelof van der Meer. Vadim Khasdan and Maria Sapojnik are contributed equally to this work. V. Khasdan · M. Sapojnik · A. Zaritsky · R. Manasherob · E. Ben-Dov Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Be’er-Sheva 84105, Israel V. Khasdan · A. R. Horowitz · M. Rippa Department of Entomology, ARO, Gilat Research Center, M.P. Negev 85280, Israel S. Boussiba Microalgal Biotechnology Laboratory, Blaustein Institute for Desert Research, Ben-Gurion University at Sede-Boker, Sede-Boker 84990, Israel R. Manasherob Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA E. Ben-Dov Achva Academic College, MP Shikmim 79800, Israel E. Ben-Dov (&) National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Be’er-Sheva 84105, Israel e-mail: [email protected]
combinations of the genes under control of an early T7, PA1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC50 of 0.27 £ 108 cells g¡1). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC50 of 0.12 £ 108 cells ml¡1) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC50 of 0.16 £ 108 cells ml¡1). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but signiWcantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, PA1 and PpsbA (constitutive), displayed signiWcantly higher toxicities (7.5- to 140-fold) than their counterparts with PA1 alone, reducing the LC50 values to below 107 cells ml¡1. Keywords Bacillus thuringiensis · -Endotoxin · Transgenic Escherichia coli · Agriculture pests
Introduction Various subspecies of the entomopathogenic bacterium Bacillus thuringiensis are speciWc and environmentally safe microbial control agents against larvae of lepidopteran, di
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