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Bt insecticide toxin coupled with abamectin and application thereof

A technology of avermectin and insecticidal toxin, applied in the field of Bt insecticidal toxin, can solve problems such as destruction, insect death, and sharp decline in control effect, and achieve simple and convenient process, good application prospect, and good insecticidal effect Effect

Active Publication Date: 2017-12-15
福建省农业科学院农业生物资源研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is generally believed that after insects feed on crystal proteins, they digest them into active toxin proteins through their own midgut proteases, and the toxin proteins bind to receptors on the brush border membranes (BBMVs) of midgut epithelial cells, and further Inserted into the membrane, forming holes or ion channels, causing ion leakage, destroying the epithelial cells of the intestinal wall, causing lysates in the intestinal tract to penetrate into the blood cavity, causing sepsis, and killing insects; however, the resistance of insects makes the existing anti-Bt toxin Therefore, it is urgent for practitioners to transform the Bt toxin and provide a Bt toxin with strong toxicity.

Method used

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  • Bt insecticide toxin coupled with abamectin and application thereof
  • Bt insecticide toxin coupled with abamectin and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Preparation of 4”-O-succinoyl abamectin

[0017] Weigh Abamectin, tert-butyldimethylsilyl chloride and imidazole according to the molar mass ratio of 1:3:6, and dissolve them in tetrahydrofuran, stir and react at room temperature for 2 hours, so as to carry out the 5-OH of Abamectin Protection; the abamectin after protection is dissolved in dichloromethane, and 4-dimethylaminopyridine, triethylamine and succinic anhydride are added, and the avermectin after protection, 4-dimethylaminopyridine, three The molar ratio of ethylamine to succinic anhydride is 1:4:8:16, after which the water bath is refluxed in a dark place for 3 hours, the abamectin 4"-OH is carboxylated, the product is extracted with ether, and the product is separated by column chromatography; Dissolve the abamectin after chromatographic separation in methanol, add p-toluenesulfonic acid, and the molar ratio of avermectin after chromatographic separation to p-toluenesulfonic acid is 1:6, and stir the reacti...

Embodiment 2

[0025] Preparation of Bacillus thuringiensis Insecticidal Protein Bt Cry2Ab Insecticidal Protein

[0026] (1) Transform the cry2Ab gene into E. coli engineering bacteria by heat shock method to obtain the E. coli engineering bacteria containing the cry2Ab gene, and then inoculate the E. coli engineering bacteria containing the cry2Ab gene on LB solid medium, and culture the LB solid The base was incubated at 30°C for 24 hours to activate the strains;

[0027] (2) Inoculate the activated Escherichia coli engineering strains in LB liquid medium, and place them in shake flasks for fermentation at 37°C, set the rotation speed at 180r / min, and wait until the bacterial liquid OD 600 After reaching 0.5, adjust the temperature to 25°C and continue to cultivate at 180r / min for 24h; then put the fermentation broth obtained from the fermentation culture at 4°C and 10000r / min and centrifuge for 10min. After the centrifugation, take the precipitate and resuspend it in Na 2 CO 3 The lysat...

Embodiment 3

[0030] Preparation of Bt insecticidal toxin

[0031] Take the 4"-O-succinoyl abamectin (0.5mmol) prepared in Example 1, dissolve it in 1mL DMSO, add EDC (0.75mmol) and NHS (0.75mmol), stir at room temperature for 2h, to 4"- The carboxyl group of O-succinoyl avermectin is activated to obtain activated 4"-O-succinoyl abamectin, which is ready for use; the Bt Cry2Ab insecticidal protein prepared in Example 2 is used to prepare 5mmol / L Bt Cry2Ab insecticidal protein Na 2 CO 3 / NaHCO 3 The buffer solution, namely the BtCry2Ab insecticidal protein solution, is ready for use; then, at 4°C, add 100 μL of activated 4”-O-succinoyl abamectin into 1 mL of the BtCry2Ab insecticidal protein solution, and stir for 1 hour A coupling reaction is carried out to obtain the Bt insecticidal toxin.

[0032] It should be noted that abamectin is a class of sixteen-membered macrolide compounds with bactericidal, insecticidal, acaricidal and nematicidal activities; after coupling of abamectin and B...

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Abstract

The invention provides a Bt insecticide toxin coupled with abamectin. The Bt insecticide toxin is formed by bonding the Bt insecticide protein Cry2Ab and 4'-O-succinoyl abamectin under the combined action of coupling agents EDC and NHS. The Bt insecticide toxin has higher toxicity, has higher insecticidal efficacy to plutella xylostella, has wider application prospect for preventing and controlling agricultural pests and can be used as an agricultural pesticide for plutella xylostella. A preparation method for the Bt insecticide toxin is simple and convenient in process and is high in operability.

Description

【Technical field】 [0001] The invention belongs to the field of agricultural pest control, and in particular relates to an abamectin-coupled Bt insecticidal toxin and its application. 【Background technique】 [0002] In recent years, with the rapid development of agricultural science, pesticide control has played a certain role in promoting. Pesticides can effectively prevent and control agricultural diseases, insects, weeds, rats and other harmful organisms, ensuring an important link in increasing agricultural production and harvest. However, the emergence of disease resistance is an unavoidable problem regardless of chemical pesticides or biological pesticides; pest resistance to pesticides is to overcome the role of pesticide sites, making them ineffective and passivated. In practical applications, the rotation application of pesticides, the mixed application of pesticides, the increase of the contact area of ​​pesticides to pests, the cultivation of sensitive strains of ...

Claims

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Application Information

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IPC IPC(8): C07K14/325C07K1/107A01P7/04
CPCC07K14/325C07K19/00
Inventor 潘志针朱育菁许炼刘波陈峥张静傅南雁
Owner 福建省农业科学院农业生物资源研究所
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