Caracterizacion Molecular E Insecticida De Nuevas Proteinas Vip De Bacillus Thuringiensis

AGL2009-13340-C02-02

Nombre agencia financiadora Ministerio de Ciencia e Innovación
Acrónimo agencia financiadora MICINN
Programa Programa Nacional de Investigación Fundamental
Subprograma Investigación fundamental no-orientada
Convocatoria Investigación fundamental no-orientada
Año convocatoria 2009
Unidad de gestión Subdirección General de Proyectos de Investigación
Centro beneficiario UNIVERSIDAD PÚBLICA DE NAVARRA (UPNA)
Centro realización ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
Identificador persistente http://dx.doi.org/10.13039/501100004837

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Draft genome sequences of two bacillus thuringiensis strains and characterization of a putative 41.9-kDa insecticidal toxin

Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
  • 0000-0002-4817-7580
  • 0000-0002-9728-1456
  • Berry, Colin
  • 0000-0001-6585-2421
  • 0000-0003-0065-9625
In this work, we report the genome sequencing of two Bacillus thuringiensis
strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic
to many lepidopteran pest species and to some mosquitoes, encoded genes for two
insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein
(Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins
(cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene
(vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both
strains, whose sequences exhibited 100% nucleotide identity. The predicted protein
showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain
(US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus,
respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was
expressed in Escherichia coli and tested for the first time against four lepidopteran species
(Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the
green-peach aphid Myzus persicae at doses as high as 4.8 μg/cm2 and 1.5 mg/mL,
respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no
mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be
toxic. While the use of the polymerase chain reaction has allowed a significant increase in
the number of Bt insecticidal genes characterized to date, novel NGS technologies promise
a much faster, cheaper and efficient screening of Bt pesticidal proteins., This research was supported by the Spanish Ministry of Science and Innovation (grant
ref. AGL2009-13340-C02) and by the Universidad Pública de Navarra (PhD contract awarded to L.P.).