A kind of Bacillus thuringiensis and its application in controlling lepidopteran pests
A technology of Bacillus thuringiensis and Bacillus aureus, applied in the direction of application, bacteria, insecticides, etc., can solve the problems of increased insecticide resistance and monotony of pests, and achieve the goal of improving the effect of insect resistance, expanding the scope of use, and improving the biological control effect Effect
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Embodiment 1
[0023] Example 1 Isolation, screening and identification of Bacillus thuringiensis CS72-2
[0024] 1. Separation and screening
[0025] The soil was collected from the plateau area of Guizhou Province and separated by sodium acetate-antibiotic separation method. The specific process is: weigh 10g of soil sample and put it into a shaker flask filled with 50ml of sodium acetate medium, add penicillin sodium salt and genta sulfate respectively 400 μg / ml of each mycin, cultured on a shaker (200 r / min, 30° C.) for 4 hours. After the cultivation, take 10ml of soil suspension, put it into a sterile centrifuge tube and centrifuge at 3000r / min for 15min, take 2ml of the upper cloudy solution and place it in a water bath at 65°C for 15min, take 0.1ml of the heat-treated cloudy solution and spread it on a plate, and place the plate in a 30°C incubator After 48 hours of culture, a smear of Bt-like strains was picked from the plate, and a Bt strain with rhomboid parasporal crystal morph...
Embodiment 2
[0030] Preliminary Identification of Example 2 Bactericidal Genes
[0031]Design a pair of specific primers based on the conserved sequence of the cyt2 gene to identify the cyt gene in strain CS72-2; the designed primer sequence is:
[0032] F: 5'-ATTTGAGTTTCTAAATTTGTAAA-3' (SEQ ID No: 2);
[0033] R: 5'-TTTACAAATTTAGAAACTCAAAT-3' (SEQ ID No: 3).
[0034] The PCR amplification system is: 0.2 μL of Taq enzyme, 2.5 μL of 10×Buffer, 1 μL of forward and reverse primers (10 μM), 5 μL of template (DNA), 2 μL of dNTP (2.5 mM), 1.5 μL of MgCl2 (25 mM), sterile Make up to 25 μL with ddH2O.
[0035] The PCR reaction program was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 90 s, 34 cycles; finally, extension at 72°C for 7 min.
[0036] The PCR products were analyzed by 1% agarose gel electrophoresis, and the results were as follows: image 3 shown by image 3 It can be known that the above-mentioned specific pr...
Embodiment 3
[0037] Example 3 Cloning and sequence analysis of the full-length gene of the bactericidal gene cyt2-like
[0038] The genomic DNA of the strain CS72-2 was extracted using a genomic DNA purification kit (purchased from Saibaisheng Company), and the cyt2-like whole was amplified using the genomic DNA as a template and cyt2-like-F and cyt2-like-R as primers. long genes.
[0039] cyt2-like-F: 5'-ATGGAGAATAAAAATCAACAC-3' (SEQ ID No: 4);
[0040] cyt2-like-R: 5'-CTATTCCTCCATAAGGAG-3' (SEQ ID No: 5).
[0041] PCR amplification system: Taq enzyme 0.2 μL, 10×Buffer 2.5 μL, forward primer (cyt2-like-F, 10 μM) 1 μL, reverse primer (cyt2-like-R, 10 μM) 1 μL, template (DNA) 5 μL , dNTP (2.5mM) 2μL, MgCl2 (25mM) 1.5μl, sterile ddH2O to make up to 25μL.
[0042] The PCR reaction program was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 90 s, 34 cycles; finally, extension at 72°C for 7 min.
[0043] The obtained PCR ...
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