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Bacillus thuringiensis fh21, insecticidal gene, expressed protein and application thereof

A technology of Bacillus thuringiensis and Bacillus aureus, applied in application, pesticide, genetic engineering and other directions, can solve the problems of increasing pesticide resistance and singleness of pests, and achieve the goal of expanding insecticidal spectrum, wide application prospects and delaying drug resistance. Effect

Active Publication Date: 2018-03-06
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the current commercialized transgenic insect-resistant crops have relatively single types of insect-resistant genes, such large-scale promotion of planting has the risk of reducing pest refuges and increasing pest resistance.

Method used

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  • Bacillus thuringiensis fh21, insecticidal gene, expressed protein and application thereof
  • Bacillus thuringiensis fh21, insecticidal gene, expressed protein and application thereof
  • Bacillus thuringiensis fh21, insecticidal gene, expressed protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, isolate Bacillus thuringiensis bacterial strain FH21

[0054] The applicant's laboratory staff obtained a strain of Bacillus thuringiensis isolated from the soil of Fenghuang Mountain in Heilongjiang Province. The spores of Bacillus thuringiensis are spore outer wall, spore coat, cortex, spore inner wall, protoplasmic membrane and protoplast from outside to inside. . The main component of the cortex is peptidoglycan, a polysaccharide teichoic acid that does not contain vegetative cells, which maintains the dehydration state and heat resistance of the spores. On the other hand, during the formation of the spores, a large amount of DPA-Ca will be produced. thuringiensis spores will not die after heat treatment at 80°C for 20 minutes, and the dormant spores are treated at a sub-lethal temperature of 75°C for 15 minutes, the activation effect is the best , not only promote its rapid germination, but also improve the survival rate of spores (Yu Ziniu 1990). A...

Embodiment 2

[0071] Example 2. Obtaining new genes

[0072] 21 Use cry gene universal primers to detect strain FH21, the primers are as follows

[0073]

[0074]

[0075] Amplification cycle: denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes.

[0076] The result is as image 3 As shown, strain FH21 was subjected to genotype PCR identification, and PCR products of different sizes were obtained by using cry1 gene identification primers.

[0077] 2.2 A rapid cloning method was used to isolate and clone the new sip1A gene in the strain.

[0078] Using pfuDNA polymerase, PCR amplification was performed with the following system.

[0079]

[0080]

[0081] Make up to 50 μL with ultrapure water, mix well and centrifuge.

[0082] Amplification cycle: denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 1 minute, 25 cycles, and finally extensi...

Embodiment 3

[0099] Embodiment 3, gene expression and activity assay

[0100] 3.1.1 Plasmid DNA was extracted from the above clones, and transformed into the recipient strain Rosetta (DE3) to obtain an expression strain.

[0101] After IPTG induced expression, SDS-PAGE protein electrophoresis was performed. The process of inducing expression is as follows:

[0102] 1) Activated strains (37°C, 12hr);

[0103] 2) 10% inoculated in LB medium (37°C, 2hr);

[0104] 3) Add the inducer IPTG, 150rpm, and induce at 18-22°C for 4-20h at low temperature;

[0105] 4) The cells were collected by centrifugation, and 10 mM Tris Cl (pH 8.0) was added to suspend;

[0106] 5) Broken bacteria (ultrasonic crushing is complete);

[0107] Centrifuge at 12,000rpm for 10min at 4°C;

[0108] Collect 10-15 μL each of the supernatant and the precipitate, and detect them by electrophoresis.

[0109] The polyacrylamide gel configuration is as follows.

[0110]

[0111] Sample loading: 10-15μl sample loading...

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Abstract

The invention relates to "bacillus thuringiensis FH21, insecticidal gene, expressed protein and application thereof", belonging to the technical field of biological control. A strain of Bacillus thuringiensis FH21, the preservation number of which is CGMCC No.10090, four insecticidal proteins were isolated from the strain, having amino acid sequences as shown in SEQ ID NO 2, 4, 6, and 8, and encoding the insecticidal protein The gene of insect protein, preferably its nucleotide sequence is shown in SEQ ID NO1, 3, 5, 7. The bacterial strain and the above-mentioned genes have high toxicity to lepidopteran pests, and can be used to transform microorganisms and plants, so that they can show toxicity to related pests, and overcome and delay the development of pesticide resistance of pests to engineering bacteria and transgenic plants.

Description

technical field [0001] The invention relates to the technical field of biological control, in particular, the invention further relates to a Bt insecticidal gene with high toxicity to lepidopteran agricultural pests and a protein encoded by the gene. Background technique [0002] Bacillus thuringiensis (Bt) is a widely distributed Gram-positive bacterium, an entomopathogenic microorganism with strong toxicity to pests and no toxicity to natural enemies, and no toxicity to higher animals and humans. It is currently the most in-depth study and the most widely used microbial insecticide, and it is active against more than 3000 kinds of pests in 16 orders. Bt can form insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs), also known as δ-endotoxin (delta-endotoxin) during the sporulation stage, its shape, structure and size are closely related to its virulence [Schnepf.E, Crickmore .N,Van Rie.J.,Lereclus.D,Baum.J,Feitelson.J,Zeigler.D.R.,Dean.D.H.Bacillus thuringi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/32C12N15/63C07K14/325A01N47/44A01P7/04C12R1/07
CPCA01N47/44C07K14/325C12N1/205C12R2001/075
Inventor 高继国李海涛刘荣梅张杰
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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