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Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests

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Abstract

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC50 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC50) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 μg/ml, respectively.

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References

  • Agrants H, Lereclus D (1991) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032

    Google Scholar 

  • Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119

    Article  CAS  Google Scholar 

  • Baum JA, Malvar T (1995) Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol Microbiol 18:1–12

    Article  CAS  Google Scholar 

  • Boonserm P, Angsuthanasombat C, Lescar J (2004) Crystallization and preliminary crystallographic study of the functional form of the Bacillus thuringiensis mosquito-larvicidal Cry4Aa mutant toxin. Acta Cryst D60:1315–1318

    CAS  Google Scholar 

  • Cowley R, Fernandez F, Freemantle W, Rutter D (1992) Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting. J Clin Microbiol 30:2451–2455

    Article  CAS  Google Scholar 

  • de Maagd RA, Bosch D, Stiekema W (1999) Bacillus thuringiensis toxin-mediated insect resistance in plants. Trends Plant Sci 4:9–13

    Article  Google Scholar 

  • de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 4:193–199

    Article  Google Scholar 

  • De Souza MT, Lecadet MM, Lereclus D (1993) Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription. J Bacteriol 175:2952–2960

    Article  Google Scholar 

  • Finney DJ (1971) Probit analysis. Cambridge Univ. Press, Cambridge UK

    Google Scholar 

  • Glare TR, O’Callaghan M (2000) Bacillus thuringiensis Biology, Ecology and Safty. Wiley, Chichester, UK

    Google Scholar 

  • Hofmann C, Lüthy P, Hütter R, Pliska V (1988) Binding of the delta-endotoxin from Bacillus thuringiensis to brush-border membrane vesicles of the cabbage butterfly (Pieris brassicae). Eur J Biochem 173:85–91

    Article  CAS  Google Scholar 

  • Kalman S, Kiehne KL, Cooper N, Reynoso MS, Yamamoto T (1995) Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes. Appl Environ Microbiol 61:3063–3068

    Article  CAS  Google Scholar 

  • Kaur S (2000) Molecular approaches towards development of novel Bacillus thuringiensis biopesticides. World J Microbiol Biotechnol 16:781–793

    Article  CAS  Google Scholar 

  • Knowles BH (1994) Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxins. Adv Insect Physiol 24:275–308

    Article  CAS  Google Scholar 

  • Knowles BH, Ellar DJ (1987) Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxin with different insect specificity. Biochim Biophys Acta 924:509–518

    Article  CAS  Google Scholar 

  • Kuo WS, Chak KF (1996) Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl Environ Microbiol 62:1369–1377

    Article  CAS  Google Scholar 

  • Lawley TD, Taylor DE (2003) Characterization of the double-partitioning modules of R27: correlating plasmid stability with plasmid localization. J Bacteriol 185:3060–3067

    Article  CAS  Google Scholar 

  • Lecadet MM, Chaufaux J, Ribier J, Lereclus D (1992) Construction of novel Bacillus thuringiensis strains with different insecticidal activities by transduction and transformation. Appl Environ Microbiol 58:840–849

    Article  CAS  Google Scholar 

  • Lereclus D, Arantes O, Chaufaux J, Lecadet MM (1989) Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60:211–218

    CAS  Google Scholar 

  • Lereclus D, Vallade M, Chaufaux J, Arantes O, Rambaud S (1992) Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination. Biotechnology 10:418–421

    Article  CAS  Google Scholar 

  • Malvar T, Gawron-Burke C, Baum JA (1994) Overexpression of Bacillus thuringiensis HknA, a histidine protein kinase homolog, bypasses early Spo− mutations that result in CryIIIA overproduction. J Bacteriol 176:4742–4749

    Article  CAS  Google Scholar 

  • Navon A (2000) Bacillus thuringiensis insecticide in crop protection-reality and prospects. Crop Prot 19:669–676

    Article  Google Scholar 

  • Park HW, Bideshi DK, Federici BA (2003) Recombinant Strain of Bacillus thuringiensis producing Cyt1A, Cry11B, and the Bacillus sphaericus binary toxin. Appl Environ Microbiol 2:1331–1334

    Article  CAS  Google Scholar 

  • Park HW, Delecluse A, Federici BA (2001) Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B. J Invertebr Pathol 78:37–44

    Article  CAS  Google Scholar 

  • Roberts L (2002) Mosquitoes and disease. Science 298:82–83

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Schnepf E, Crickmore N, van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    Article  CAS  Google Scholar 

  • Sekar V (1988) The insecticidal crystal protein gene is expressed in vegetative cells of Bacillus thuringiensis var. tenebrionis. Curr Microbiol 17:347–349

    Article  CAS  Google Scholar 

  • Song FP, Zhang J, Huang DF, Xie TJ, Dai LY, Li GX (1998) Establishment of PCR-RFLP identification system of cry genes from Bacillus thuringiensis. Sci Agric Sin 31:19–24

    Google Scholar 

  • Song FP, Zhang J, Gu AX, Wu YE, Han LL, He KL, Chen ZY, Yao J, Hu YQ, Li GX, Huang DF (2003) Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-Type gene. Appl Environ Microbiol 69:5207–5211

    Article  CAS  Google Scholar 

  • Tabashnik BE, Finson N, Chilcutt CF, Cushing NL, Johnson MW (1993) Increasing efficiency of bioassays: evaluation of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J Econ Entomol 86:635–644

    Article  Google Scholar 

  • Whalon ME, Wingerd BA (2003) Bt: mode of action and use. Arch Insect Biochem Physiol 54:200–211

    Article  CAS  Google Scholar 

  • Yu JX, Pang Y, Tang MJ, Xie RY, Tan L, Zeng SL, Yuan MJ, Liu JY (2001) Highly toxic and broad-spectrum insecticidal Bacillus thuringiensis engineered by using the transposon Tn917 and protoplast fusion. Curr Microbiol 43:112–119

    Article  CAS  Google Scholar 

  • Zhang J, Song FP, Li CY, Chen ZY, Tan JX, Huang DF (2002) Cloning and expression of cry3Aa7 gene from Bacillus thuringiensis strain toxic to Coleopteran pests. Sci Agric Sin 35(6):650–653

    CAS  Google Scholar 

Download references

Acknowledgements

We are very grateful to Ms. Yingping Liang and Dr. Gemei Liang for test larvae supply and help in bioassays. We thank Dr. Didier Lereclus, in Pateur Institute, France, Dr. Yiping Wang in Peking University, China and Ming Sun, in Huazhong Agricultural University, Wuhan of China for the experiment material. This work was supported by 863 Plan of China (No.2004AA214091, No.2002AA212061) and 973 Projects of China (No.2001CB109005, No.2003CB114201).

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Authors and Affiliations

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, People’s Republic of China

    Guangjun Wang, Jie Zhang, Fuping Song & Jun Wu

  2. Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Baoding 071000, People’s Republic of China

    Shuliang Feng

  3. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China

    Dafang Huang

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  1. Guangjun Wang
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  2. Jie Zhang
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  3. Fuping Song
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  4. Jun Wu
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  5. Shuliang Feng
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  6. Dafang Huang
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Correspondence to Jie Zhang or Dafang Huang.

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Wang, G., Zhang, J., Song, F. et al. Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests. Appl Microbiol Biotechnol 72, 924–930 (2006). https://doi.org/10.1007/s00253-006-0390-x

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  • DOI: https://doi.org/10.1007/s00253-006-0390-x

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