High-throughput insecticide screening method for targeting insect Kir1 channel and application of high-throughput insecticide screening method

A screening method and high-throughput technology, applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc., can solve few problems and achieve the effects of fast detection speed, high specificity and reliability

Active Publication Date: 2017-12-29
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the many insecticide target proteins, only the high-throughput screening technology for insecticides targeting acetylcholinesterase is relatively successful, but the types of insecticides (organophosphates and carbamates) targeting acetylcholinesterase have been around for more than 60 years Due to its activity and safety issues, there are very few companies paying attention to this type of insecticide

Method used

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  • High-throughput insecticide screening method for targeting insect Kir1 channel and application of high-throughput insecticide screening method
  • High-throughput insecticide screening method for targeting insect Kir1 channel and application of high-throughput insecticide screening method
  • High-throughput insecticide screening method for targeting insect Kir1 channel and application of high-throughput insecticide screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of the inward rectifier potassium channel gene Kir1 in brown planthopper and beet armyworm

[0030] RNA extraction kits were used to extract total RNA from N. lugens and Spodoptera lugens, and cDNA was obtained by reverse transcription. Using the cDNA as a template, specific primers (SEQ ID NO.1: 5'-ATGGAAGCGGCCACCAGA-3 ', SEQ ID NO.2:5'-CTAAACCAGGCTCGTATTG-3') and the specific primer (SEQ ID NO.3:5'-ATGTCAGGATTAAAAAGATGTAT-3', SEQ ID NO.4:5'-TCACTTTTCTCATTTACTGTGCTC -3') for PCR amplification, the reaction system includes 12.5 μL 2×Taq Mix, 1 μL each of the upstream and downstream primers, add sterilized ultrapure water to a total volume of 25 μL; the amplification program is: 94°C pre-denaturation for 3 minutes, 94°C denaturation 30s, renaturation at 55°C for 30s, extension at 72°C for 2min, after 30 cycles, total extension at 72°C for 10min. After recovery, the PCR product was ligated with the pMD-19T vector, and the ligated product was transforme...

Embodiment 2

[0031] Example 2: Construction of Kir1 expression plasmid

[0032] The expression vector pcDNA3 was amplified to prepare its plasmid, and the pcDNA3 plasmid was double-digested with restriction endonucleases (Apal and Kpnl) to obtain a linear pcDNA3 vector. The Kir1 sequences with Apal and Kpnl restriction sites at both ends were connected into the pcDNA3 vector, and then transformed into Escherichia coli, positive clones were selected, and the resulting pcDNA3-NlKir1 and pcDNA3-SeKir1 expression plasmids were sequenced and verified.

Embodiment 3

[0033] Example three: transfected cells

[0034] The pcDNA3-NlKir1 and pcDNA3-SeKir1 plasmids were transfected into HEK293 cells. Before the transfection test, HEK293 cells in the logarithmic growth phase were digested with trypsin, inoculated in a 24-well culture plate, added complete medium and placed in a CO2 incubator. When the cells grew to 60-80%, take Opti -MEM Medium Wash the cells and replace the complete medium in the dish. The configuration of the transfection mixture: Take three 1.5mL EP tubes and add 200 μL of Opti-MEM medium and 1-2 μg of purified pEGFP-N1-SeKir1, pEGFP-N1-NlKir1, pEGFP-N1 (blank vector) plasmids, Take another 3 1.5mL EP tubes, add 200 μL of Opti-MEM medium and 2 μL of Lipofectin 2000 reagent, let it stand for about 5 minutes, gently mix the Opti-MEM medium containing the carrier and transfection reagent to form liposome-DNA complexes After standing at room temperature for 5-30 minutes, slowly add the liposome-DNA mixture to the culture plate, ...

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Abstract

The invention discloses gene sequences of inwardly rectifying potassium channels Kir1 of brown rice planthopper and beet armyworm and a high-throughput insecticide screening method established by using the channels. The genes Kir1 of the inwardly rectifying potassium channels disclosed by the invention are obtained by cloning hemipterous insects and lepidoptera insects for the first time; the Kir1 channels are closely related to regulation and control of physiological processes such as herbivory and excretion of insects; and the inhibition of the channels possibly affects the herbivory and excretion process of the insects or even causes the death of the insects, and therefore, the channels can be developed as a novel insecticide target. The Kir1 genes are integrated into an expression vector, are expressed in cells by transfection and are continuously screened to obtain a monocolonal cell line for stably expressing the Kir1; the monocolonal cell line can be used for stably expressing the Kir1 channels with the inwardly rectifying characteristic; and the influence of a tested compound on potassium fluxes of the Kir1 channels is determined by using a thallium fluorescent reagent according to the equivalence of thallium ions and potassium ions passing through the Kir1 channels, and further a potential insecticide having inhibitory activity for the Kir1 channels are screened from a plurality of candidate compounds.

Description

technical field [0001] The invention belongs to the field of plant protection and biotechnology, and in particular relates to an insect inward rectification potassium channel and a screening method using the channel as an insecticide target. Background technique [0002] Agricultural pests cause extremely serious economic losses to crops every year, and it is necessary to use pesticides to control the damage to ensure the safety of food and economic crops. However, the irrational use of pesticides has gradually led to adaptive evolution of the pest population, and the emergence of drug-resistant populations in the field has reduced the control effect of pesticides, and even failed to control them. This requires continuous research and development of new types of pesticides to replace existing ones Severely resistant varieties. There are many types of insecticides currently used in production, such as organophosphates, carbamates, nereistoxins, pyrethroids, neonicotinoids, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/85G01N21/64
CPCC07K14/43563C12N15/85G01N21/6428G01N21/6486
Inventor 苏建亚任淼淼
Owner NANJING AGRICULTURAL UNIVERSITY
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