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Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae

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Abstract

The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), which has previously been shown to encode the cry8Ga toxin gene, is active against both Holotrichia oblita and Holotrichia parallela. Recombinant Cry8Ga however is only weakly toxic to these insect pests suggesting the involvement of additional toxins in the native strain. We report that through the use of Illumina sequencing three additional, and novel, genes, namely vip1Ad1, vip2Ag1, and cry8-like, were identified in this strain. Although no protein corresponding to these genes could be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the HBF-18 proteome, reverse transcription (RT)-PCR indicated that all three genes were transcribed in the native strain. The two vip genes were cloned and expressed and, as with other Vip1/2 toxins, appeared to function as a binary toxin and showed strong activity against H. oblita, H. parallela and Anomala corpulenta. This is the first report to demonstrate that the Vip1/Vip2 binary toxin is active against these Scarabaeoidea larvae. The cry8-like gene appeared to be a C-terminally truncated form of a typical cry8 gene and was not expressed in our usual recombinant Bt expression system. When however the missing C-terminal region was replaced with the corresponding sequence from cry8Ea, the resulting hybrid expressed well and the toxin was active against the three test insects.

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Acknowledgments

This work was partially funded by National High Technology Research and Development Project (863 Project, Grant No. 2011AA10A203) and the National Transgenic Major Program (Grant No. 2014ZX08009-013B). We would like to thank Ms. Guihua Chen for the help with the bioassays.

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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, 100193, China

    Yang Bi, Yanrui Zhang, Changlong Shu, Fuping Song & Jie Zhang

  2. School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK

    Neil Crickmore

  3. Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou, 061001, China

    Qinglei Wang

  4. Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Baoding, 071000, China

    Lixin Du

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  1. Yang Bi
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  2. Yanrui Zhang
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  3. Changlong Shu
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  4. Neil Crickmore
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  5. Qinglei Wang
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  6. Lixin Du
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  7. Fuping Song
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  8. Jie Zhang
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Corresponding author

Correspondence to Jie Zhang.

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Yang Bi and Yanrui Zhang contributed equally to this work.

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Bi, Y., Zhang, Y., Shu, C. et al. Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae. Appl Microbiol Biotechnol 99, 753–760 (2015). https://doi.org/10.1007/s00253-014-5966-2

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