Synthetic genes encoding CRY1AC
A coding, nucleotide sequence technology, applied in genetic engineering, recombinant DNA technology, peptides, etc., can solve problems such as insufficient control of heliothine
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Embodiment 1
[0089] Example 1. Synthetic Nucleotide Sequence Encoding Cry1Ac
[0090] A synthetic nucleotide sequence (SEQ ID NO: 6) encoding Cry1Ac was designed. These sequences are represented herein by SEQ ID NO: 1 (synFLCry1Ac), SEQ ID NO: 2 (synCry1Ac-variant 1), SEQ ID NO: 3 (synCry1AcB), SEQ ID NO: 4 (synCry1AcC) and SEQ ID NO: 5 ( optCry1Acv02) said.
Embodiment 2
[0091] Example 2. Expression of synthetic cry1Ac sequences in Bacillus
[0092] The synthetic cry1Ac sequence was amplified by PCR and cloned into the Bacillus expression vector pAX916 by methods well known in the art. After transformation into cells of the cry(-) Bacillus thuringiensis strain, the resulting clones were assayed for expression of the Cry1Ac protein. Bacillus strains comprising the synCry1Ac clone and expressing the Cry1Ac insecticidal protein are grown, for example, in CYS medium (10 g / l Bacto-casitone; 3 g / l yeast extract; 6 g / l KH 2 PO 4 ;14g / l K 2 HPO 4 ; 0.5mM MgSO 4 ; 0.05mM MnCl 2 ;0.05mM FeSO 4 ) until sporulation was evident by microscopic examination. Samples were prepared and analyzed by polyacrylamide gel electrophoresis (PAGE).
Embodiment 3
[0093] Example 3. Determination of insecticidal activity
[0094] The ability of the synthetic cry1Ac nucleotide sequence of the present invention to produce an insecticidal protein was detected. The ability of pesticidal proteins to act as pesticides against pests is often assessed in a number of ways. One method known in the art is to perform feeding assays. In this feeding assay, pests are exposed to a sample containing the compound to be tested or to a control sample. Typically, this is done by placing the material to be tested, or a suitable dilution of such material, on a substance that the pest will ingest, such as an artificial diet. The material to be tested can consist of liquids, solids or slurries. The material to be tested can be placed on the surface and allowed to dry. Alternatively, the material to be tested can be mixed with a molten artificial diet and dispensed into the assay chamber. Assay chambers can be, for example, cups, dishes, or wells of a mic...
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