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Determination method for evaluating toxicity on lepidopterous larvae by Bt toxic protein

A lepidopteran larvae and assay method technology, applied in the field of assaying the toxicity of Bt toxins to lepidopteran larvae, achieving the effects of high efficiency, low test cost, and accurate assay

Inactive Publication Date: 2016-08-17
INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for evaluating the toxicity of Bt toxic protein to Lepidoptera larvae. This method can prevent the protein from being degraded at high temperature, the concentration is mixed uniformly and the waste of protein, and the economic cost is low. Simple, accurate determination of Bt toxin virulence

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  • Determination method for evaluating toxicity on lepidopterous larvae by Bt toxic protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Assessing method for evaluating the toxicity of Cry1F toxic protein to the larvae of cutworm

[0017] (1) Preparation of artificial feed for cutworm larvae: prepare the artificial feed for small cutworm larvae according to the method described in CN 200810112399.7, when the prepared artificial feed is cooled to 50° C. (before solidification), pour into a plastic squeeze bottle, Then pour it into a 24-well plate, where 1.5-2ml is squeezed into each small hole, and the diameter of the hole is 1.6cm. After solidification, the surface area of ​​the feed is 2cm. 2 ,spare;

[0018] (2) Preparation of test objects: newly hatched larvae within 12 hours after hatching of cutworms;

[0019] (3) Bt toxic protein staining and toxicity measurement on the feed surface: the Cry1F protein to be tested is diluted serially into 10 concentrations (0, 1.3, 2.6, 5.2, 10.4, 20.8) with CAPs buffer (pH=10.5) , 41.6, 83.3, 166.6, 333.3 μg / ml), use a pipette gun to draw 40 μl of ...

Embodiment 2

[0022] Example 2: Evaluation of the toxicity assay method of Cry1F toxic protein to newly hatched larvae of corn borer

[0023] (1) Preparation of corn borer artificial feed: prepare corn borer artificial feed according to the method described in CN 97100519.2, when the prepared artificial feed is cooled to 50°C (before solidification), pour it into a plastic squeeze bottle, and then squeeze it into 24 In the orifice plate, squeeze 1.5-2ml into each small hole, the diameter of the hole is 1.6cm, after solidification, the surface area of ​​the feed is 2cm 2 ,spare.

[0024] (2) Test object preparation: newly hatched larvae within 12 hours after hatching of corn borer.

[0025] (3) Bt toxic protein coating on feed surface and the mensuration of toxicity: the Cry1F protein to be tested is buffered with CAPs, pH=10.5, carry out serial dilution into 7 concentrations (0, 0.03125, 0.0625, 0.125, 0.25, 0.5 , 1, 2 μg / ml), use a pipette gun to draw 40 μl of protein liquid of each conc...

Embodiment 3

[0028] Example 3: Evaluation of the toxicity assay method of Cry1F toxic protein to newly hatched armyworm larvae

[0029](1) Preparation of artificial feed for armyworm: prepare artificial feed for armyworm according to the method described in CN201010197333. Put it into a 24-well plate, squeeze 1.5-2ml into each small hole, the diameter of the hole is 1.6cm, after solidification, the surface area of ​​the feed is 2cm 2 ,spare.

[0030] (2) Test object preparation: newly hatched larvae within 12 hours after hatching of armyworm.

[0031] (3) Bt toxic protein staining on feed surface and mensuration of toxicity: the Cry1F protein to be tested is buffered with CAPs, pH=10.5, carry out serial dilution into 9 concentrations (0, 1.3, 2.6, 5.2, 10.4, 20.8 , 41.6, 83.3, 166.6 μg / ml), use a pipette gun to draw 40 μl of protein liquid of each concentration into the small hole, shake well, so that the protein completely covers the surface of the feed, and finally make the concentrati...

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Abstract

A method for evaluating the toxicity of Bt toxins to Lepidoptera larvae, comprising the following steps: (1) artificial feed pretreatment; (2) Bt toxins on the surface of the feed and the measurement of toxicity; (3) testing statistics. The method for measuring Bt protein toxicity of the present invention adopts squeeze bottle feed and pours it into a 24-well culture plate, which is fast and efficient, and can form a uniform surface area. After adding Bt protein, a uniform and stable surface concentration can be formed, which can not only accurately Measuring the toxicity of Bt protein can also save protein and achieve the effect of lower testing cost.

Description

technical field [0001] The invention relates to a method for evaluating the toxicity of Bt toxic protein, in particular to a method for evaluating the toxicity of Bt toxic protein to Lepidoptera larvae. Background technique [0002] Bacillus thuringiensis (Bt) is a kind of crystal-producing bacillus that includes many subspecies and is highly virulent to a variety of insects. It belongs to Gram-positive bacteria and is the most widely used microbial insecticide in the world. . When sensitive insects ingest Bt crystal protein, parasporal crystals enter the midgut, undergo dissolution and activation under alkaline conditions, release toxin core fragments, and then act on the epithelial cells of the midgut, causing the cells to swell and lyse, and finally lead to The insect gut is paralyzed, perforated, or stops feeding until the body dies. Therefore, there is a need for a suitable virulence assay in terms of evaluating the virulence of Bt insecticides or Bt toxins, differenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 李国平封洪强黄建荣钟景封洪云黄博田彩红邱峰
Owner INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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