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Bacillus bombyseptieus parasporal crystal toxin (Bb toxin) protein and truncated segment thereof as well as recombinant expression vector and application of Bacillus bombyseptieus parasporal crystal toxin protein

A technology of bacillus and paraspore crystals, applied in the field of biochemistry, can solve the problem of few reports on the pathogenic mechanism

Inactive Publication Date: 2014-03-12
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Bacillus bombyseptieus (Bb) is a widely distributed Gram-positive bacterium that is highly pathogenic to insects, but there are few reports on its pathogenic mechanism

Method used

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  • Bacillus bombyseptieus parasporal crystal toxin (Bb toxin) protein and truncated segment thereof as well as recombinant expression vector and application of Bacillus bombyseptieus parasporal crystal toxin protein
  • Bacillus bombyseptieus parasporal crystal toxin (Bb toxin) protein and truncated segment thereof as well as recombinant expression vector and application of Bacillus bombyseptieus parasporal crystal toxin protein
  • Bacillus bombyseptieus parasporal crystal toxin (Bb toxin) protein and truncated segment thereof as well as recombinant expression vector and application of Bacillus bombyseptieus parasporal crystal toxin protein

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1, Separation and purification, activation and mass spectrometric identification of Bb toxin

[0026] According to the separation and purification method of Bacillus thuringiensis parasporal crystal toxin protein (Bt Cry1Ac), the Bacillus nigerosepticum parasporal crystal toxin protein (Bb toxin) was purified, specifically: Bb bacteria were inoculated in 5 mL of LB medium (pancreatic 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.2) in a test tube, 30°C, 250r / min shaking overnight; inoculate 1% volume in LB medium, 30°C, 250r / min Cultivate for about 34-38 hours. From 34 hours, take samples to observe the production of paraspore crystals and spores with oil immersion; centrifuge the bacterial solution at 4°C and 7000rpm for 15 minutes; wash with pre-cooled 1M NaCl to remove impurities in the medium Centrifuge the protein at 4°C, 8000rpm for 15 minutes (repeat twice); wash twice with pre-cooled sterile water; add 100mL lysate (50mM Na 2 CO 3 , 25mM EDTA, add 5%-m...

Embodiment 2

[0032] Example 2, Bb toxin cloning, prokaryotic expression and expression and purification of truncated fragments thereof

[0033] Specific primers were designed according to the Bb genome sequence, the forward primer of Bb toxin: 5'-ccatatggtgagtctgaaaaagaaattagg-3' (SEQ ID NO.2), and the reverse primer 5'-ccctcgagttatttttcttccccagctt-3' (SEQ ID NO.3). Using Bb genomic DNA as a template for amplification, the PCR reaction conditions are: 95°C pre-denaturation for 5 minutes, then 95°C denaturation for 40 seconds, 54°C annealing for 40 seconds, 72°C extension for 50 minutes, a total of 28 cycles, and finally 72°C extension 10 minutes; the PCR product was identified and recovered by agarose gel electrophoresis, and then ligated with the pMD19-T Simple vector. The ligation reaction was performed under the action of Solution I ligase at 16°C for 2 h, and then transformed into DH5α competent cells to obtain The positive clone pMD19-T Simple / Bb toxin was sent to Huada Gene Company f...

Embodiment 3

[0037] Example 3 Lepidopteran insect Bombyx mori adds food to the survival rate detection of Bb toxin truncated fragments

[0038] Take the silkworm eggs of the Lepidoptera insect Bombyx mori DZ and soak them in acid (the specific gravity of hydrochloric acid is 1.073, the temperature is 46°C, and the time is 5 minutes) to relieve diapause, and put them in a dark environment with 15°C and 85% humidity for about 30 days. Until hatching, the ant silkworms were harvested and placed in a standard environment (temperature: 25°C, humidity: 80%) and raised with mulberry leaves until the 4th instar.

[0039] After the 4th instar silkworm larvae were starved for 6 hours, the truncated Bb toxin parasporal crystal toxin protein was fed orally at 60ug / head, and 2 repeated areas were set up, with 40 silkworms in each repeated area, and 2 silkworms were fed with the same volume at the same time The 4th instar silkworm in PBS was used as the control area, and the survival rate and death rate...

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Abstract

The invention discloses Bacillus bombyseptieus parasporal crystal toxin (Bb toxin) protein and a truncated segment thereof as well as a recombinant expression vector and an application of the Bacillus bombyseptieus parasporal crystal toxin protein. The amino acid sequence of the Bacillus bombyseptieus parasporal crystal toxin protein is shown by SEQ ID NO.4, and the truncated segment of the Bacillus bombyseptieus parasporal crystal toxin protein is shown by SEQ ID NO.8. The survival rate of domestic silkworms is obviously reduced (about 30%) after silkworm larvae are additionally fed with the truncated segment, which shows that the gene can be used for coding a toxic protein capable of causing lepidopteron, namely the domestic silkworms to death, and the Bacillus bombyseptieus parasporal crystal toxin protein can be used for preparing biological pesticides for resisting lepidoptera pests and has wide application prospect.

Description

technical field [0001] The present invention belongs to the field of biochemistry, and specifically relates to Bb toxin (Bb toxin) and a truncated fragment of Bb toxin containing Bb toxin, and also relates to Bb toxin containing Bb toxin. Recombinant expression vector and application of spore crystal toxin protein. Background technique [0002] Bacillus thuringiensis parasporal crystal toxin protein (Bt Cry) has insecticidal activity against Lepidoptera, Diptera, Coleoptera, Hymenoptera and other insects. After the Bt Cry gene was introduced into tobacco and tomato and showed insect-resistant properties, insect-resistant transgenic corn, rice, potato, cabbage, cotton, poplar, etc. were successively obtained. Taking my country's genetically modified cotton as an example, the direct economic benefits brought by it each year reach 15 billion yuan, increase income and reduce expenditure by about 150 yuan per mu, and reduce the amount of chemical pesticides by 20-30 million kilo...

Claims

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Application Information

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IPC IPC(8): C07K14/32C12N15/32C12N15/70C12N1/21A01N43/50A01P7/04
CPCA01N43/50C07K14/32
Inventor 程廷才林平金盛凯常怀谱夏庆友
Owner SOUTHWEST UNIVERSITY
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