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Application of Chouioia cunea Yang cytochrome p450CYP4C3 gene in insecticide stress resistance

A Zhou's gnawing wasp and insecticide technology, applied in the field of genetic engineering, can solve the problems of pesticide residues, affecting the natural control effect of wasps, etc., and achieve the effect of avoiding self-injury

Active Publication Date: 2022-04-15
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the release of the white moth Chow's gnats can effectively control the American white moth, but in the wild environment, the vitality of the wasps will be affected by multiple factors, among which, the pesticide residue is an important environment that affects the natural control effect of the wasps factor

Method used

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  • Application of Chouioia cunea Yang cytochrome p450CYP4C3 gene in insecticide stress resistance
  • Application of Chouioia cunea Yang cytochrome p450CYP4C3 gene in insecticide stress resistance
  • Application of Chouioia cunea Yang cytochrome p450CYP4C3 gene in insecticide stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Evaluation of Toxicity of Insecticides on A.

[0029] Toxicity evaluation of insecticides against the white moth Chowlii was carried out using the film method, and different insecticides were diluted to effective concentrations of 0, 100, 200, 300, 400, 500 mg / L (diflubenzuron) , 0, 3, 6, 9, 12, 15 mg / L (beta-cypermethrin), 0, 10, 20, 30, 40, 50 mg / L (matrine · nicotine). Fill a clean 50 mL wide-mouthed Erlenmeyer flask with liquid medicine, let it stand for 5 minutes, pour out the liquid medicine in the tube, and let it dry naturally, so that the medicine liquid evenly forms a film on the wall of the tube. There were 50 bees, and the mouth of the bottle was tightly sealed with 120-mesh mesh cloth. After 6 hours, the number of dead bees in the bottle was checked and counted.

Embodiment 2

[0031] Obtained the CYP4C3 gene sequence of the white moth A. zhoui

[0032] The experimental materials were obtained from the indoor subculture of the white moth A. zhouzii (cultivation conditions: in an artificial climate box (PQX-350H), temperature 25°C, relative humidity 60%-80%, no light and dark). and collected into RNase-free centrifuge tubes (1.5ml) as the control group; sublethal concentrations (LC10, LC30) of diflubenzuron, beta-cypermethrin, and matrine·nicotine were used to stress the white moth Zhou's gnats respectively. After 6 hours, 200 surviving individuals were selected, completely submerged in RNA later, and collected into RNase-free centrifuge tubes (1.5ml) as the experimental group. Three replicates were set up for each group of samples, stored overnight in a refrigerator at 4°C, and transferred to an ultra-low temperature refrigerator the next day. After the sample preparation was completed, it was sent to Biomic Biotechnology Company for transcriptome s...

Embodiment 3

[0035] Extraction of total RNA

[0036] Distilled water was used as a control, and the 1-day-old adult bee was treated with sublethal concentrations (LC10, LC30) of diflubenzuron, beta-cypermethrin, and matrine and nicotine by the drug film method for 6 hours. After collecting the surviving individuals, the product was used immediately or stored at -20°C and frozen.

[0037] (1) Take about 30 of the white moth Chow's gnats into a 1.5mL sterile centrifuge tube, pour liquid nitrogen into it and grind it quickly with a grinding rod. Transfer the powdered sample to a centrifuge tube, add 1mL RNA isolater, and let it stand; after the sample is completely melted, continue pipetting until the lysate is transparent.

[0038] (2) Transfer the lysate to a centrifuge tube, centrifuge at 12,000 g at 4°C for 5 min, and aspirate the supernatant.

[0039] (3) Add 1 / 5 volume of chloroform (200 μL) to the above lysate. Shake vigorously for 15 sec to form an emulsion, and stand at 4°C for 5 ...

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Abstract

The invention discloses a cDNA (complementary deoxyribonucleic acid) full-length sequence of a natural enemy insect Chouioia cunea Yang p450 enzyme system CYP4C3 gene and application of the cDNA full-length sequence in insecticide stress resistance, the full length of the gene sequence is 1521 bp, and the gene sequence contains a complete open reading frame and codes 506 amino acids. Real-time fluorescent quantitative PCR (polymerase chain reaction) experiments find that the expression quantity of CYP4C3 genes of Chouioia cunea Yang can be increased when insecticides such as chlorbenzuron, beta-cypermethrin and nicotine-sophocarpidine for chemical prevention and control of fall webworms are subjected to stress treatment, and particularly, the CYP4C3 gene expression can be obviously increased when the stress of the concentration of an insecticide (LC30) for inducing death of 30% of Chouioia cunea Yang is applied to stress treatment of insecticides such as chlorbenzuron, beta-cypermethrin and nicotine-sophocarpidine. The invention can provide a molecular basis for coordinating chemical control and biological control of fall webworms.

Description

[0001] This invention was funded by the National Natural Science Foundation of China (31702058) and Tianjin Higher Education Science and Technology Development Project (2019KJ089). technical field [0002] The invention belongs to the technical field of genetic engineering and relates to the study of the p450 enzyme system of the white moth A. chowii CYP4C3 The full-length cDNA sequence and different sublethal concentrations of insecticide stress CYP4C3 Changes in gene expression. Background technique [0003] American White Moth ( Hyphantria cunea Drury) is one of the important diseases and insect pests that seriously threaten the agricultural and forestry production in my country. At present, chemical agents are mostly used for the control of American white moth. However, improper use of chemical insecticides will not only pollute the environment, but may also lead to continuous drug resistance Enhance, but also kill natural enemies, reduce their long-term natural contro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12Q1/6888
Inventor 潘丽娜金欣高雯芳胡鹏飞刘新宇覃东玉任睿张天天李敏
Owner TIANJIN NORMAL UNIVERSITY
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