Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene
A Bacillus thuringiensis and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of increasing insecticide resistance and singleness of pests, and achieve the effects of expanding insect spectrum, wide application prospects, and delaying insecticide resistance
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Embodiment 1
[0036] Embodiment 1, isolate Bacillus thuringiensis bacterial strain LB-R-78
[0037]The applicant's laboratory staff obtained a strain of Bacillus thuringiensis from the soil near Lalin Town, Wuchang City, Heilongjiang Province. membranes and protoplasts. The main component of the cortex is peptidoglycan, a polysaccharide teichoic acid that does not contain vegetative cells, which maintains the dehydration state and heat resistance of the spores. On the other hand, during the formation of the spores, a large amount of DPA-Ca will be produced. thuringiensis spores will not die after heat treatment at 80°C for 20 minutes, and the dormant spores are treated at a sub-lethal temperature of 75°C for 15 minutes, the activation effect is the best , not only promote its rapid germination, but also improve the survival rate of spores (Yu Ziniu 1990). According to this characteristic, temperature screening can be implemented (Knowles B H, Ellar D J. Colloid-osmotic lysis is a general ...
Embodiment 2
[0053] Example 2. Obtaining new genes
[0054] 2.1 Use cry1 gene universal primers to detect strain LB-R-78, the primers are as follows
[0055]
[0056]
[0057] Amplification cycle: denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes.
[0058] The result is as image 3 As shown, strain LB-R-78 was identified by PCR-RFLP genotype, and a PCR product with a size of 1.6 kb was obtained by using cry1 gene identification primer K5un2 / K3un2, which was subjected to Pst I / Xba I double enzyme digestion analysis, The RFLP map was obtained. It can be seen from this figure that the amplified 1.6kb PCR product produced 9 restriction fragments after enzyme digestion, the sizes were 801bp, 758bp, 518bp, 423bp, 322bp, 239bp, 140bp, 95bp, 16bp (Kuo et al, 1996), the results showed that: LB-R-78 contains cry1Ca and cry1Ac genes.
[0059] 2. Cloning of cry1Ca gene in 1LB-R-78 strain ...
Embodiment 3
[0107] Embodiment 3, gene expression and activity assay
[0108] 3.1.1 Plasmid DNA was extracted from the above clones, and transformed into the recipient strain Rosetta (DE3) to obtain an expression strain.
[0109] After IPTG induced expression, SDS-PAGE protein electrophoresis was performed.
[0110] The process of inducing expression is as follows:
[0111] 1) Activated strains (37°C, 12hr);
[0112] 2) 10% inoculated in LB medium (37°C, 2hr);
[0113] 3) Add the inducer IPTG, 150rpm, and induce at 18-22°C for 4-20h at low temperature;
[0114] 4) The cells were collected by centrifugation, and 10 mM Tris Cl (pH 8.0) was added to suspend;
[0115] 5) Broken bacteria (ultrasonic crushing is complete);
[0116] Centrifuge at 12,000rpm for 10min at 4°C;
[0117] Collect 10-15 μL each of the supernatant and the precipitate, and detect them by electrophoresis.
[0118] The polyacrylamide gel configuration is as follows.
[0119]
[0120] Sample loading: 10-15μl sampl...
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