Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis
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The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding δ-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 combinations of the genes under control of an early T7, P A1 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 significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC50 values to below 107 cells ml−1.
KeywordsBacillus thuringiensis δ-Endotoxin Transgenic Escherichia coli Agriculture pests
The authors thank Michael Davidovich, Svetlana Kontsedalov and Sophia Kleitman from the Agricultural Research Organization (ARO), and Monica Einav of BGU’s Life Sciences Department, for their technical assistance. Aaron Leeper from the ARO, Gilat is gratefully acknowledged for his valuable editorial comments. This investigation was partially supported by grants of the Chief Scientist of the Israel Ministry of Agriculture (No. 857-0481-03 to AZ, RH and SB) and from the United States-Israel Binational Science Foundation (B.S.F.), Jerusalem, Israel (AZ and SB) (No. 2001-042).
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