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New pesticide synergistic technology and application thereof

A gene and cytochrome technology is applied in the field of pesticide synergy in the regulation of the transcription of pest detoxification enzyme genes, and the inhibition of the transcription of pest detoxification enzyme genes, which can solve the problem that resistant pests have no cure, cannot solve the problem of multi-drug resistance, and reduce the Detoxification and metabolism of harmful organisms, etc., to achieve a wide range of synergistic effects

Pending Publication Date: 2021-06-29
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the phenomenon of multi-drug resistance, in which the pests are resistant to multiple pesticides, is quite common, resulting in the situation that these resistant pests have no cure
Although the use of synergists is also one of the resistance control measures, such as synergistic ether can reduce the detoxification and metabolism of harmful organisms by inhibiting the activity of cytochrome P450 detoxification enzymes, and has a certain synergistic effect on resistant pests. Efficacy agents can only target a certain type of detoxification enzymes, and the pest populations with multidrug resistance are often caused by the increased activity of multiple detoxification enzymes. Therefore, traditional synergistic technologies cannot solve the problem of multidrug production. resistance problem

Method used

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  • New pesticide synergistic technology and application thereof
  • New pesticide synergistic technology and application thereof
  • New pesticide synergistic technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Cloning of the upstream regulatory sequence of the CYP gene of beet armyworm and brown planthopper

[0041] RNA extraction kits were used to extract total RNA from brown planthopper and beet armyworm respectively, and cDNA was obtained by reverse transcription, and then using cDNA as a template, PCR amplification was performed using primers for the upstream regulatory sequence of the CYP gene, and the reaction system included 12.5 μL 2× 1 μL each of Taq Mix and upstream and downstream primers, add ddH 2 0 to 25 μL. The amplification program was: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and after 30 cycles, extension at 72°C for 10 min. After PCR product recovery and pMD 19 -T vector ligation, the ligation product was transformed into Escherichia coli DH 5α competent cells, positive clones were screened by blue and white spots, positive clones were sent for sequencing for veri...

Embodiment 2

[0042] Example 2: Construction of a fluorescent reporter plasmid

[0043] Select Xhol I and hind III restriction sites on the fluorescent reporter plasmid PGL3-Basic, and perform double digestion to linearize them. The double restriction reaction system includes: Xhol I 1 μL, Hind III 1 μL, 10×fast Cut buffer 5 μL, PGL3-Basic plasmid 1 μg, make up to 50 μL with double distilled water, and then react at 37°C for about 2 hours to obtain a linearized plasmid. Design specific primers according to the instruction manual of CloneExpress II One Step Cloning Kit, and add homologous sequences at the end of the linearized PGL3 vector to the 5' end of the original specific primers, that is, two restriction site sequences of Xhol I and hind III to form a new the amplification primers. Then, the CYP gene upstream regulatory sequence (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 cloned in embodiment one , SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO....

Embodiment 3

[0044] Example three: transfected cells

[0045] Place Sf9 cells in a biochemical incubator at 27°C for culture, add fetal bovine serum with a final content of 5% to the SF9-900 II SFM medium as the cell culture medium, and passage the cells every three days to ensure the viability of the cells. Sf9 cells cultured for about 2 days and whose viability was above 95% were used for the experiment. Gently aspirate the culture medium in the culture dish, use a pipette gun to draw 1mL of fresh medium containing 5% serum, gently blow and suspend the adherent cells, and transfer the blown cells to a 50mL centrifuge tube, take 10 μL of cell solution was thoroughly mixed with 10 μL of Coomassie Brilliant Blue and added to a cell counting plate for cell counting. Calculate the amount of cells to be added according to the cell counting results, and reduce the cell concentration in the centrifuge tube to 8×10 5 A / mL or so. In a 24-well cell plate, add 500 μL per well containing 4×10 5 T...

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Abstract

The invention provides a novel pesticide synergistic technology and application thereof, and discloses upstream regulatory sequences of beet armyworm and brown planthopper P450 genes and a transcription inhibitor screening method established by using the upstream regulatory sequences. Metabolic resistance of pests to insecticides is caused by genomic up-regulation expression of detoxification enzymes such as P450, the up-regulation expression is controlled by the upstream regulatory sequences of the up-regulation expression, transcription of detoxification genes can be down-regulated by inhibiting transcriptional activity of the regulatory sequences, and the control effect of the insecticides is improved. The upstream regulatory sequences are responsible for up-regulation expression of the resistance-related P450 gene. The regulatory sequences are correspondingly integrated into fluorescent reporter plasmids, the fluorescent reporter plasmids are expressed in cells through transfection, and the intensity of a fluorescent signal is controlled by an insertion sequence. When an inhibitor is added into transfected cells, the transcription of the regulatory sequences on luciferase can be influenced, so that the intensity of a fluorescence signal is influenced. By utilizing the novel pesticide synergistic technology, an insecticidal synergist for inhibiting the transcription of the detoxification enzyme genes can be screened from numerous candidate compounds.

Description

technical field [0001] The invention belongs to the field of plant protection and biotechnology, and specifically relates to the transcription regulation of pest detoxification enzyme gene and the pesticide synergistic technology for inhibiting the transcription of pest detoxification enzyme gene. [0002] technical background [0003] The use of pesticides in agricultural production is the most important measure to prevent and control crop diseases, insect pests and weeds. However, the repeated use of pesticides for a long time puts continuous screening pressure on farmland pests, resulting in adaptive micro-evolution of pests, and the evolution of pests to produce resistance. The control effect of pesticides will be reduced, or even the control will fail, which will seriously affect the control effect of pests and the sustainability of agricultural production, and even threaten food security and people's health. Therefore, the control of pest resistance is a serious practic...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/65C12Q1/6888
CPCC12N9/0081C12N15/65C12Q1/6888C12Q2600/124C12Q2600/158C12Y114/15006
Inventor 苏建亚胡波胡松竹
Owner NANJING AGRICULTURAL UNIVERSITY
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