Insecticidal polypeptides having broad spectrum activity and uses thereof
A technology of insecticidal activity and polypeptide sequence, which is applied in the fields of peptide, application, and decapeptide
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Embodiment 1
[0249] Example 1 - Gene Identification and Escherichia coli (E.coli) Expression
[0250]The gene encoding the insecticidal protein of SEQ ID NO: 4 can be obtained from a Bacillus thuringiensis strain designated BD380. The selected Bt strains were grown in 30 mL of medium with shaking at 250 rpm for 16 hours. Isolated DNA was collected from cells using a kit from Macherey-Nagel (Catalog #740521). DNA was then sequenced, assembled and annotated using the Illumina HiSeq2500. Then select the gene of interest.
[0251] The coding sequence of SEQ ID NO: 3 for the insecticidal polypeptide of SEQ ID NO: 4 was synthesized and cloned into pET28a vector and transformed into E. coli BL21 cells (Invitrogen). Large scale 1.0 L cultures were grown until O.D. 600nm~0.8, then the cultures were induced with isopropyl β-D-1-thiogalactoside (IPTG) 1 mM and grown at 16°C for 16 hours. Cell pellets were lysed with 50 mL of 500 mM NaCl / 20 mM Tris / 5 mM imidazole / pH 7.9 supplemented with 0.02%...
Embodiment 2
[0252] Example 2 - Lepidoptera Assay Using Partially Purified Proteins
[0253] Insecticidal Activity Bioassay Screens to Evaluate the Effects of Insecticidal Proteins on Various Lepidopteran Species: European Corn Borer (Ostrinia nubilalis), Corn Earworm (Helicoverpa zea), Lesser Cutworm (Agrotisipsilon), Fall Armyworm (Spodoptera frugiperda), soybean looper (Pseudoplusia includesens) and bean armyworm (Anticarsia gemmatalis).
[0254] Lepidoptera feeding assays were performed on artificial diets containing purified protein in 96-well plate devices. Purified protein (25ul) was then added to artificial diet. Put 2 to 5 newborn larvae into each hole, and feed them arbitrarily for 5 days. Results are indicated as positive for larval responses such as stunting and / or death. The result was indicated as negative if the larvae were similar to the negative control fed a diet supplemented with the above buffer only. On European corn borer (Ostrinia nubilalis), corn earworm (Heli...
Embodiment 3
[0258] Example 3 - Agrobacterium-mediated transformation of maize and regeneration of transgenic plants
[0259] For carrying out Agrobacterium-mediated maize transformation with a polynucleotide sequence (such as SEQ ID NO: 1 or SEQ ID NO: 3), the method of Zhao (US Patent No. 5,981,840 and PCT Patent Publication WO98 / 32326; The contents of the patent are incorporated herein by reference). Briefly, immature embryos are isolated from maize, and the embryos are suspended with Agrobacterium in which the bacterium is capable of transferring a toxin nucleotide sequence (for example, SEQ ID NO: 1 or SEQ ID NO: 3) to at least one unripe embryo. The contacting is carried out in the condition of at least one cell of the mature embryo (step 1: infection step). In this step, immature embryos can be dipped into the Agrobacterium suspension to prime the inoculation. The embryos are co-cultivated with Agrobacterium for a period of time (step 2: co-cultivation step). After this infecti...
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