A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains
The #cry# gene content of #Bacillus thuringiensis# subsp. #aizawai# HD-133 was analyzed by a combination of highpressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the #cry#1 family (#cry#1Aa, #cry#1Ab, #cry#1C, and #cry#1D) as well as a gene each from the #cry#2 (#cry#2B) and the #cry#1l families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, #Cry#1Ab, #Cry#1C, and #Cry#1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the #cry#2B and #cry#1I genes, but not the #cry#1Aa gene, were transcribed. Cloning and sequencing of the #cry#1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of #B. thuringiensis# isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total #cry# gene content of #B. thuringiensis# strains may be higher than previously thought.
| Main Authors: | , , , , , |
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| Format: | article biblioteca |
| Language: | eng |
| Published: |
American Society for Microbiology
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| Subjects: | H10 - Ravageurs des plantes, Bacillus thuringiensis, pouvoir pathogène, bactérie entomopathogène, biotechnologie, génie génétique, résistance aux organismes nuisibles, http://aims.fao.org/aos/agrovoc/c_761, http://aims.fao.org/aos/agrovoc/c_5629, http://aims.fao.org/aos/agrovoc/c_34819, http://aims.fao.org/aos/agrovoc/c_16165, http://aims.fao.org/aos/agrovoc/c_15974, http://aims.fao.org/aos/agrovoc/c_5731, |
| Online Access: | http://agritrop.cirad.fr/401138/ http://agritrop.cirad.fr/401138/1/401138.pdf |
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| Summary: | The #cry# gene content of #Bacillus thuringiensis# subsp. #aizawai# HD-133 was analyzed by a combination of highpressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the #cry#1 family (#cry#1Aa, #cry#1Ab, #cry#1C, and #cry#1D) as well as a gene each from the #cry#2 (#cry#2B) and the #cry#1l families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, #Cry#1Ab, #Cry#1C, and #Cry#1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the #cry#2B and #cry#1I genes, but not the #cry#1Aa gene, were transcribed. Cloning and sequencing of the #cry#1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of #B. thuringiensis# isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total #cry# gene content of #B. thuringiensis# strains may be higher than previously thought. |
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