Bt protein Cry2Ac-like and coding gene and application thereof
A technology of bt protein and protein, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of reducing usage, reducing cost, and improving insecticidal activity
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Embodiment 1c
[0034] Cloning of embodiment 1cry2Ac-like gene
[0035] The new bacterial strain JF21-1 of Bacillus thuringiensis (Bacillusthuringiensis) that the present invention isolates from the soil of Muchuan primeval forest area, Sichuan Province, this bacterial strain has been on August 11, 2011 in the General Microorganism Center of China Microbiological Culture Collection Management Committee (address : No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) is preserved, classified as Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCCNo.5119.
[0036] In this example, the full-length sequence of the cry2Ac-like gene was cloned by the following method.
[0037] Genomic DNA Purification Kit (purchased from Saibaisheng Company) was used to extract the total DNA of strain JF21-1 as a template for amplifying the cry2Ac-like gene, and the primer sequences were design...
Embodiment 2
[0043] Example 2 Obtaining of Cry2Ac-like protein
[0044] According to the sequence at both ends of the open reading frame of the cry2Ac-like gene, a pair of specific primers L2AcF: 5′-CGGAGCTCATGACGATGAAAACAAGGCA-3′, L2AcR: 5′-ATTTGCGGCCGCCTATAGAAATGGGAGTTACA-3′ were designed and synthesized, and the bases underlined at the 5′ end primers were SacI and NotI restriction sites. The total DNA of JF21-1 was used as a template to amplify, and the digested product was ligated with the vector pET-28a(+) after the same double digestion, and transformed into E.coliDH5α competent cells, and its plasmid was extracted and electrophoresed to verify the inserted fragment After the size fits the intended purpose fragment ( figure 2 ), and then transferred into the recipient bacteria E.coli.BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant plasmid was named pET-2L, and the recombinant containing the recombinant plasmid was named E.coli.BL21(2L). Cul...
Embodiment 3
[0046] Example 3 Determination of Cry2Ac-like protein insecticidal activity
[0047] The Cry2Ac-like protein obtained in Example 2 was tested for its insecticidal activity against Aedes mosquitoes. First, the Cry2Ac-like protein was formulated into 6 different concentration gradients of 0.1, 4, 8, 16, 32, 64mg / mL, and then 20 Aedes larvae were put into each treatment, and each treatment was repeated 3 times, E.coli.BL21 (DE3) and empty vector pET-28a(+) were used as negative controls, and clear water was used as blank control; statistical results after 12 hours, LC 50 Analyzed with SPSS10.0 software.
[0048] The result shows (table 1): expression product has certain insecticidal activity to Aedes mosquito, LC 50 It was 36.54 μg / mL. The bioassay results showed that E.coli.BL21(DE3), empty vector pET-28a(+), and blank control had no insecticidal activity against Aedes mosquitoes.
[0049] Table 1 Insecticidal activity of Cry2Ac-like against Aedes mosquitoes
[0050]
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