Pesticidal gene and its use
An insecticidal gene, gene technology, applied in genetic engineering, plant genetic improvement, application, etc.
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Embodiment 1
[0012] Embodiment 1, the clone acquisition of gene and synthetic gene:
[0013] The steps of gene cloning are as follows:
[0014] 1. Take 300 different Bt strains and culture them with TB culture solution at 28°C for 24 hours, then mix all the culture solutions, according to the method of Reddy et al. J. Bacteriol.169, 5263-5270) to extract the plasmid of Bt. This plasmid is a mixture of plasmids from 300 different Bt strains.
[0015] 2. Using the above-mentioned Bt plasmid as template DNA, PCR amplification was carried out with primers StartN 5'atg aac aag aat aat act aaatta agc aca aga and StopC 5'cat gtt act cgaa ttc tcg ag tta. A total of 30 cycles of PCR amplification were performed. The first five cycles are 95°C, 30 seconds; 50°C, 60 seconds; 72°C, 2 minutes 30 seconds, followed by 25 cycles: 95°C, 30 seconds; 60°C, 60 seconds; 72°C, 2 minutes 30 seconds Second.
[0016] 3. The PCR product was analyzed by electrophoresis, and the DNA of about 2.4 kb was recovered...
Embodiment 2
[0018] Embodiment 2, the preparation of albumen:
[0019] The expression vector pET28a-LG01 containing the above genes was transformed into Escherichia coli BL21star by a general standard method, and a colony containing the plasmid pET28a-LG01 was obtained. Inoculate a single colony into 100 ml of LB bacterial culture medium, shake culture at 37°C to OD 600 =0.6, add IPTG to 0.5mM, and continue to culture under the same conditions for 4 hours. The culture solution was centrifuged at 5000 g for 10 minutes to pellet E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 ml of 20mM Tris-HCl buffer solution to the precipitate and ultrasonically pulverize it for the determination of insecticidal activity.
Embodiment 3
[0020] Example 3, the protein expressed in Escherichia coli has killing effect on various Lepidoptera insects:
[0021] The protein obtained in Example 2 was coated on the surface of the artificial diet for insects, and after standing for 2 hours, the newborn first-instar larvae were connected to measure the insecticidal activity. The preparation method of the negative control was the same as in Example 2, but the plasmid was the pET28a vector itself without any inserted DNA. Under the condition of 25°C, record the number of insects killed after 7 days. The insecticidal activity results are as follows:
[0022] Armyworm
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