Insecticide toxin protein HS487 and application of gene thereof in biological prevention and treatment

A technology of insecticidal toxins and proteins, applied in the direction of microorganism-based methods, applications, biocides, etc., to achieve the effects of wide insecticidal spectrum, high insecticidal activity, and high insecticidal efficiency

Active Publication Date: 2016-08-31
NANJING AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, chemical pesticides are also having an increasing negative impact on the environment

Method used

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  • Insecticide toxin protein HS487 and application of gene thereof in biological prevention and treatment
  • Insecticide toxin protein HS487 and application of gene thereof in biological prevention and treatment
  • Insecticide toxin protein HS487 and application of gene thereof in biological prevention and treatment

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Cloning of insecticidal toxin protein hs487 gene

[0033] Ⅰ. Extraction of total DNA from Serratia nematophila R187-2 (the strain preservation number is CCTCC M 2016240):

[0034] A. Take 1.5mL of the expanded cultured R187-2 bacterial solution in a sterilized centrifuge tube, mark, centrifuge at 6000rpm / min for 10min, discard the supernatant, and keep the bacterial pellet.

[0035]B. Add 1mL ddH to the centrifuge tube 2 In O, after vortexing evenly, centrifuge at 6000rpm / min for 10min, discard the supernatant, and keep the bacterial pellet.

[0036] C. Add 1mL ddH to the centrifuge tube again 2 O, after vortexing evenly, a concentrated bacterial solution was obtained.

[0037] D. Take 100 μL concentrated bacterial solution into a 1.5 mL sterilized centrifuge tube, add 668 μL TE and 32 μL 50 mg / ml lysozyme solution, mix thoroughly, and place in a 37-degree constant temperature water bath for 2 hours.

[0038] E. After that, add 200 μL of 10% SDS, shake...

Embodiment 2

[0101] The preparation method of preparing the insecticidal toxin protein HS487: under the condition of 37°C and 200rpm, cultivate the BL21(DE3) strain containing the pET-32a(+)-hs487 recombinant plasmid overnight in the LB liquid medium of 100 μg / mL Amp as a transfer seed broth. Transfer 1:1000 into LB liquid medium with a resistance of 100 μg / mL Amp, incubate at 37°C and 200 rpm for 5 hours, so that the OD600 of the bacterial liquid reaches about 0.6, take 1 mL of the bacterial liquid in a sterile 1.5 ml EP tube, Add IPTG to the remaining bacterial solution to a final concentration of 0.2mM, induce for 3 hours at 28°C at low speed (150rpm), take out the bacterial solution and place it on ice for 10 minutes, centrifuge at 4°C, 8000rpm for 15 minutes, and wash with 50mM Tris-HCl buffer (pH 7.8) Wash the cell pellet twice, after each washing, centrifuge at 4°C, 8000rpm for 10min, and collect the cell pellet.

[0102] The collected precipitate of induced bacteria was resuspende...

Embodiment 3

[0121] Detection of hemocoel toxicity of insecticidal toxin protein HS487 to the wax moth:

[0122] In order to detect the insecticidal activity of the prokaryotic-expressed insecticidal toxin protein HS487, the activity of the toxin protein HS487 crudely extracted and purified from the fermentation broth and the prokaryotic-expressed toxin protein HS487 were detected by blood cavity injection of S. mellonella moth. Dissolve the lyophilized powder of the crude extract of fermented protein and the lyophilized powder of prokaryotic expressed toxin protein HS487 with sterilized insect saline respectively, divide them into two groups, each group has an experimental group and a control group, use a microsyringe from the 5th instar 0.1 μg, 0.2 μg, 0.4 μg, 0.8 μg, 1.6 μg, and 2.0 μg of protein were injected into the blood cavity between the second pair of abdominal segments of mature larvae of S. mellonella mellonella. The larvae were placed in an incubator at 30°C, and 30 larvae wit...

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Abstract

The invention discloses insecticide toxin protein HS487. The invention also discloses an encoding gene hs487 of insecticide toxin protein. The invention also discloses a recombinant carrier and a transgene cell system or transgene recombination bacteria containing the gene hs487 of insecticide toxin protein. The invention also discloses applications of the gene hs487 of insecticide toxin protein, the recombinant expression carrier, the transgene cell system or the transgene recombinant bacteria in preparing the insecticide toxin protein HS487. The invention also discloses a preparation method of the insecticide toxin protein HS487. The invention also discloses a preparation of the insecticide toxin protein HS487 prepared by the preparation method. An amino acid sequence is induced by the encoding gene hs487 of the insecticide toxin protein of serratia nematodiphila R187-2, and the synthesized insecticide toxin protein HS487 has higher morbidity on multiple types of agricultural pests. The insecticide toxin protein has the beneficial effects that the insecticide activity is high, the insecticide spectrum is wide, and theinsecticide efficiency is high.

Description

technical field [0001] The invention relates to insecticidal toxin protein HS487 of Serratia nematodiphila strain R187-2 and its gene and application in biological control, belonging to the field of biological pesticides. Background technique [0002] In the long process of agricultural planting, chemical pesticides have played an indelible role in promoting its development. However, due to long-term and large-scale use or even abuse of pesticides, the problem of resistance has become more and more serious, which has attracted people's attention. Not only the problem of resistance, but also the pollution of agricultural and sideline products caused by chemical pesticides has also aroused people's concerns. A large number of pesticide accidents occur in our country every year, which has brought huge economic losses reaching 100 million yuan. At the same time, chemical pesticides have increasingly negative impacts on the environment. Therefore, the agricultural field has beg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/24C12N15/70A01N47/44A01P7/04C12R1/425
CPCA01N47/44C07K14/24
Inventor 张克云杨欢柳李旺周桂信林建严秀文莫国香侯祥龙阚飞徐良
Owner NANJING AGRICULTURAL UNIVERSITY
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