Novel bacillus thuringiensis gene with coleopteran activity
A technology of insecticidal activity and amino acids, applied in genetic engineering, biocides, peptides, etc.
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Embodiment 1
[0195] Example 1: Bioassay for Testing the Insecticidal Activity of Bacillus thuringiensis Toxins on Selected Insects
[0196] A bioassay was performed to evaluate the effect of the Bt insecticidal toxin peptide as described in SEQ ID NO: 2 on western corn rootworm. Feeding analysis of artificial diets containing insecticidal proteins. Topical application of insecticidal proteins using a coleopteran-specific artificial diet. Toxin was applied at a rate of 1.0 μg per 25 μl sample per container and allowed to dry. The protein was contained in 10 mM carbonate buffer at pH 10. One newborn larva was placed in each container to feed ad libitum for 5 days. The result is positive if there is a larval reaction such as shrunken and / or dead. The result is negative if the larvae are similar to the negative controls consuming the diet with only the above buffer applied.
[0197] Table 1: Results of feeding bioanalysis of SEQ ID NO: 2
[0198]
Embodiment 2
[0199] Example 2: Determining LC 50 and EC 50
[0200] Bioassays were performed to determine the LC of the insecticidal toxin peptide as described in SEQ ID NO: 2 against western corn rootworm (Maize rootworm) 50 and EC 50 . Feeding analysis of artificial diets containing insecticidal proteins. Insecticidal proteins were diluted with 10 mM carbonate buffer at pH 10 and insect diet to achieve final concentrations of toxin of 50000, 5000, 500, 50 and 5 ppm. One newborn larva was placed in each container to feed ad libitum for 5 days. At each dose, each bioassay was performed in eight replicates and the bioassay was repeated three times. Express results as LC by mortality 50 and / or express it as EC by weighing the surviving larvae at each toxin concentration 50 .
Embodiment 3
[0201] Example 3: Transformation of maize by particle bombardment and regeneration of transgenic plants
[0202] Immature maize embryos from greenhouse donor plants are bombarded with DNA molecules containing the toxin nucleotide sequence (e.g., SEQ ID NO: 1) associated with the ubiquitin promoter and selectable marker genes PAT (Wohlleben et al. (1988) Gene 70:25-37) is operably linked, which confers resistance to the bialaphos herbicide. Alternatively, selectable marker genes are provided on separate DNA molecules. The conversion is performed as follows. The medium formulation is as follows.
[0203] Preparation of target tissue
[0204] To peel the ears, use 30% CLOROX TM Bleach plus 0.5% micro-detergent to sanitize the surface for 20 minutes, rinse twice with distilled water. Cut and place the immature embryos, hypocotyl side down (cotyledon disc side up), 25 embryos per plate, place in 560Y medium for 4 hours, and then arrange them in the target area of 2.5cm for ...
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