DNA encoding insecticidal cry1bf bacillus thuringiensis proteins and recombinant hosts expressing same
A protein and protein-coding technology, applied in the field of DNA sequence, can solve problems such as amino acid sequence differences
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Embodiment 1
[0075] Example 1: Strain characteristics
[0076] The strain BtS02072BG was isolated from a dust particle sample collected by Santo Tomas la Union in Ilocos, Philippines. The strain BtS02739C was isolated from a dust particle sample collected in Lucena City, South Tagalog, Philippines.
[0077] Each strain can be cultured on conventional standard medium, preferably T 3 Medium (tryptone 3g / l, trypsin 2g / l, yeast extract 1.5g / l, 5mg MnCl 2 , 0.05MNa 2 HPO 4 ·2H 2 O, 0.05M NaH 2 PO 4 ·H 2 0, pH 6.8 and 1.5% agar), preferably cultured at 28°C. For long-term storage, it is preferable to mix an equal volume of spore crystal suspension with an equal volume of 50% glycerin and store at -70°C or freeze-dry the spore crystal suspension. In order to form spores, it is preferred to 3 Grow on the medium at 28°C for 72 hours and then store at 4°C. The crystal protein produced by the strain during sporulation is wrapped into crystals.
Embodiment 2
[0078] Example 2: Insecticidal activity of strains BtS02072BG and BtS02739C against selected lepidopteran insect species
[0079] Use the spore crystal mixture obtained from BtS02072BG or BtS02739C on artificially fed primary larvae of Helicoverpa zea, Heliothisvirescens, Ostrinia nubilalis, Spodopterafrugiperda and Sesamia nonagrioides (Approximately 10 9 Spore crystals / ml) for toxicity analysis.
[0080] The artificial feed (Vanderzant, 1962) was distributed by holes on the Costar 24-well plate to test the American cotton bollworm, tobacco budworm and corn borer. 50 microliters of spore crystal mixture was applied to the feed surface and dried in a stratified air stream. In order to test the American cotton bollworm and tobacco budworm, one larva was placed in each hole, and 20 larvae were used for each sample. In order to test the corn borer, 2 larvae were placed in each hole and 24 larvae were used for each sample. The artificial feed was distributed on the Costar 48-well plat...
Embodiment 3
[0083] Example 3: Characteristics of the new cry gene
[0084] PCR was performed using degenerate primers targeting the conserved regions in the known cry gene to detect the BtS02739C gene. Use Wizard PCR preps (Promega) purification system to purify the amplified product and connect it to pGEM-T vector (Promega). The ligation mixture was transformed into E. coli JM 101 by electroporation. A small-scale preparation consists of at least 40 transformants containing inserts and digested with selected restriction enzymes. Electrophoresis is performed on the digested small-scale DNA preparation, and then different types of DNA fragments can be observed. Choose at least one colony of each type. Prepare appropriate DNA preparations to sequence the plasmid inserts in each selected colony. The sequence of the determined amplified product and the published cry sequence indicate that the strain BtS02739C contains a new cry1J-type gene and a new cry9-type gene.
[0085] The detection of BtS02...
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