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Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

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Abstract

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.

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Literature Cited

  1. MJ Adang MJ Staver TA Rocheleau J Leighton RF Barker DV Thompson (1985) ArticleTitleCharacterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta Gene 36 289–300 Occurrence Handle10.1016/0378-1119(85)90184-2 Occurrence Handle3000881

    Article  PubMed  Google Scholar 

  2. H Arvidson PE Dunn S Strnad AI Aronson (1989) ArticleTitleSpecificity of Bacillus thuringiensis for lepidopteran larvae: Factors involved in vivo and in the structure of a purified protoxin Mol Microbiol 3 1533–1543 Occurrence Handle2615654

    PubMed  Google Scholar 

  3. HP Bietlot JP Schernthaner RE Milne FR Clairmont RS Bhella H Kaplan (1993) ArticleTitleEvidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA J Biol Chem 268 8240–8245 Occurrence Handle8463332

    PubMed  Google Scholar 

  4. BL Brizzard HR Whiteley (1988) ArticleTitleNucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. thuringiensis Nucleic Acids Res 16 2723–2724 Occurrence Handle3362680

    PubMed  Google Scholar 

  5. JA Chambers A Jelen MP Gilbert CS Jany TB Johnson C Gawron-Burke (1991) ArticleTitleIsolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai J Bacteriol 173 3966–3976 Occurrence Handle2061280

    PubMed  Google Scholar 

  6. FR Clairmont RE Milne VT Pham MB Carrière H Kaplan (1998) ArticleTitleRole of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis J Biol Chem 273 9292–9296 Occurrence Handle10.1074/jbc.273.15.9292 Occurrence Handle9535923

    Article  PubMed  Google Scholar 

  7. XZ Ding LQ Xia (2001) ArticleTitleScreening of a highly toxic insecticidal strain 4.0718 of Bacillus thuringiensis Chin J Biol Control 17 163–166

    Google Scholar 

  8. WP Donovan CC Dankocsik MP Gilbert WC Gawron-Burke RR Groat BC Carlton (1988) ArticleTitleAmino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki J Biol Chem 263 561–567 Occurrence Handle3121615

    PubMed  Google Scholar 

  9. H Höfte P Soetaert S Jansens M Peferoen (1990) ArticleTitleNucleotide sequence and deduced amino acid sequence of a new Lepidoptera-specific crystal protein gene from Bacillus thuringiensis Nucleic Acids Res 18 5545 Occurrence Handle2216728

    PubMed  Google Scholar 

  10. H Höfte HR Whiteley (1989) ArticleTitleInsecticidal crystal proteins of Bacillus thuringiensis Microbiol Rev 53 242–255 Occurrence Handle2666844

    PubMed  Google Scholar 

  11. G Honée T Salm Particlevan der B Visser (1988) ArticleTitleNucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60.5 coding for a toxin highly active against Spodoptera species Nucleic Acids Res 16 6240 Occurrence Handle3399402

    PubMed  Google Scholar 

  12. S Kalman KL Kiehne JL Libs T Yamamoto (1993) ArticleTitleCloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae Appl Environ Microbiol 59 1131–1137 Occurrence Handle8476286

    PubMed  Google Scholar 

  13. W-S Kuo K-F Chak (1996) ArticleTitleIdentification 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 Occurrence Handle8919799

    PubMed  Google Scholar 

  14. H-K Lee SS Gill (1997) ArticleTitleMolecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis Appl Environ Microbiol 63 4664–4670

    Google Scholar 

  15. D Lereclus O Arantes J Chaufaux M Lecadet (1989) ArticleTitleTransformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis FEMS Microbiol Lett 51 211–217 Occurrence Handle10.1016/0378-1097(89)90511-9 Occurrence Handle2550317

    Article  PubMed  Google Scholar 

  16. IR Pendleton RB Morrison (1966) ArticleTitleSeparation of the spores and crystals of Bacillus thuringiensis Nature 212 728–729

    Google Scholar 

  17. J Sambrook EF Fritsch T Maniatis (1989) Molecular cloning: A laboratory manual EditionNumber2 Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY

    Google Scholar 

  18. F Sanger S Nicklen AR Coulson (1977) ArticleTitleDNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci USA 74 5463–5467 Occurrence Handle271968

    PubMed  Google Scholar 

  19. J Sasaki S Asano N Hashimoto BW Lay S Hastowo H Bando T Iizuka (1997) ArticleTitleCharacterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto Curr Microbiol 35 1–8 Occurrence Handle10.1007/s002849900201 Occurrence Handle9175551

    Article  PubMed  Google Scholar 

  20. JP Schernthaner RE Milne H Kaplan (2002) ArticleTitleCharacterization of a novel insect digestive DNase with a highly alkaline pH optimun Insect Biochem Mol Biol 32 255–263 Occurrence Handle10.1016/S0965-1748(01)00084-4 Occurrence Handle11804797

    Article  PubMed  Google Scholar 

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

    Google Scholar 

  22. HE Schnepf HC Wong HR Whiteley (1985) ArticleTitleThe amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence J Biol Chem 260 6264–6272 Occurrence Handle2581950

    PubMed  Google Scholar 

  23. FP Song J Zhang DF Huang TJ Xie ZW Yang LY Dai GX Li (1998) ArticleTitleEstablishment of PCR-RFLP identification system of cry genes from Bacillus thuringiensis Sci Agric Sin 31 13–18

    Google Scholar 

  24. B Visser E Munsterman A Stoker WG Dirkse (1990) ArticleTitleA novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein J Bacteriol 172 6783–6788 Occurrence Handle2254254

    PubMed  Google Scholar 

  25. H Wabiko KC Raymond LA Bulla SuffixJr (1986) ArticleTitleBacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis DNA 5 305–314 Occurrence Handle3743328

    PubMed  Google Scholar 

  26. WR Widner HR Whiteley (1989) ArticleTitleTwo highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities J Bacteriol 171 965–974 Occurrence Handle2914879

    PubMed  Google Scholar 

  27. D Wu XL Cao YY Bai AI Aronson (1991) ArticleTitleSequence of an operon containing a novel δ-endotoxin gene from Bacillus thuringiensis FEMS Microbiol Lett 81 31–36 Occurrence Handle10.1016/0378-1097(91)90466-N

    Article  Google Scholar 

  28. Y Yokoyama K Kohda M Okamoto (1998) ArticleTitleCytA protein, a delta-endotoxin of Bacillus thuringiensis subsp. israelensis is associated with DNA Biol Pharm Bull 21 1263–1266 Occurrence Handle9881635

    PubMed  Google Scholar 

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Acknowledgments

We thank Dr. B. M. Hansen for useful suggestions and critical reading of manuscript. This investigation was supported by a NSFC grant (30270037), an 863 grant (2002AA245021), China, and a grant (KSCX2-SW-301-10) from the Chinese Academy of Sciences.

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Authors and Affiliations

  1. College of Life Science, Hunan Normal University, Changsha, 410081, PR, China

    Liqiu Xia, Yunjun Sun, Xuezhi Ding, Zujiao Fu, Xiangtao Mo & He Zhang

  2. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, PR, China

    Yunjun Sun & Zhiming Yuan

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  1. Liqiu Xia
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Correspondence to Liqiu Xia.

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Xia, L., Sun, Y., Ding, X. et al. Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis. Curr Microbiol 51, 53–58 (2005). https://doi.org/10.1007/s00284-005-4504-y

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