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Preparation of fruit flies having modified nilaparvata lugens CYP6ER1 gene, and application of fruit flies to pesticide screening

A technology of transgenic and brown planthoppers, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of complexity and inaccuracy, and achieve the effects of fast reproduction, overcoming insoluble and easy breeding

Pending Publication Date: 2020-08-11
NANJING JIXING BIOTECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In insects, the detoxification metabolism mediated by P450 monooxygenase is one of the main mechanisms for the increase of insecticide resistance, but the process of mediating insecticide resistance is highly complex. If only a simple up-regulation of P450 gene expression It is inaccurate to judge that it mediates the corresponding resistance. For example, 18 P450 genes were induced and up-regulated after imidacloprid was treated with N. lugens, but in vitro expression experiments found that only 4 of these P450 enzymes could metabolize imidacloprid. Insecticide resistance, further confirmation of function is required after finding up-regulated gene expression

Method used

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  • Preparation of fruit flies having modified nilaparvata lugens CYP6ER1 gene, and application of fruit flies to pesticide screening
  • Preparation of fruit flies having modified nilaparvata lugens CYP6ER1 gene, and application of fruit flies to pesticide screening
  • Preparation of fruit flies having modified nilaparvata lugens CYP6ER1 gene, and application of fruit flies to pesticide screening

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Calling and vector construction of CYP6ER1

[0036] In order to complete the present invention, it is first necessary to obtain the complete open reading frame (ORF) of the CYP6ER1 gene. Many studies have found that there are multiple transcripts of the CYP6ER1 gene of the brown planthopper, including CYP6ER1vA, CYP6ER1vB, CYP6ER1vC, CYP6ER1vD, CYP6ER1vF / L, etc., wherein CYP6ER1vA The transcripts of CYP6ER1vA have more powerful functions, and the inventors have also found that the transcripts that mainly exist in the field brown planthopper population in my country are also CYP6ER1vA, so the present invention selects CYP6ER1vA transcripts for the construction of transgenic fruit flies, hereinafter referred to as CYP6ER1;

[0037] First, find the nucleotide sequence of the CYP6ER1 transcript on the NCBI website, and then use the primer design website ( https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) Design a pair of amplification primers comprising the enti...

Embodiment 2

[0049] Embodiment 2: Preparation of transgenic fruit flies

[0050] (1) Inject the pJFRC2-10XUAS-IVS-mCD8::GFP-CYP6ER1 plasmid into the genotype: or In the Drosophila embryo, under the action of PhiC31 integrase, the attB sequence on the plasmid specifically recognizes the attP sequence in Drosophila, and the target gene is inserted into the Drosophila II chromosome in a single copy. The successfully transgenic Drosophila will Red compound eyes appear;

[0051] (2) The genetic background of the final fruit fly of the present invention is Drosophila melanogaster that does not contain PhiC31 integrase in the genome and is a corpus luteum removed. Therefore, it is necessary to remove the PhiC31 integrase by hybridization. Select the phenotype of corpus luteum and red eye, and then match the genotype as: or The background Drosophila single-pair hybridization, the offspring select male adults with luteum body and red eyes, and then with the balanced line Drosophila There ...

Embodiment 3

[0052] Example 3: Determination of Transgenic Drosophila Sensitivity to Insecticides and Screening of Compounds

[0053] CYP6ER1 transgenic male flies were crossed with da-Gal4 virgin flies as the experimental group, and CYP6ER1 transgenic flies were crossed with w 1118 Drosophila, da-Gal4 Drosophila and w 1118 The hybrid combination of Drosophila is a control group, and the offspring produced by hybridization are used for test determination. The experimental group contains a complete Drosophila Gla4 / UAS binary expression drive system, which does not exist in the two control groups. In the present invention, the Fluorescent quantitative PCR was used to determine the driving effect of the driving system in the offspring of the treatment group and the two control groups;

[0054] (1) First, according to the principles of qRT-PCR primer design, using the nucleotide sequence of the CYP6ER1 gene transferred into Drosophila as a template, use the online website (http: / / bioinfo.ut.e...

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Abstract

The invention relates to an application of a nilaparvata lugens CYP6ER1 gene to gene modification of fruit flies. The nilaparvata lugens CYP6ER1 gene is constructed in the fruit flies, a GAL4 / UAS binary expression system of the fruit flies is utilized, the CYP6ER1 gene can be excessively expressed in the fruit flies, the fruit flies can be used for determining whether the CYP6ER1 gene has the effects of removing toxicity and promoting metabolism on frequently-used pesticides controlling nilaparvata lugens to define whether the CYP6ER1 gene mediates resistance of the nilaparvata lugens to the pesticides, and besides, the fruit flies can be used for screening and developing novel pesticides.

Description

technical field [0001] The present invention relates to the fields of molecular biology, animal model construction and pest control thereof. Specifically, the present invention relates to constructing the P450 gene in the brown planthopper into the fruit fly, and using the GAL4 / UAS binary expression system of the fruit fly to build a rapid To determine the interaction between the CYP6ER1 gene of brown planthopper and insecticide resistance and to screen the fruit fly model for new drugs to control brown planthopper. [0002] technical background [0003] Brown planthopper (Nilaparvata lugens) is an important pest in rice production. It is widely distributed in Asian countries such as China, South Korea, Japan, and the Philippines. It not only causes direct damage by feeding on the liquid nutrients of rice, but also spreads rice straw-like dwarfism. Rice grassy stunt virus (RGSV) and rice ragged stunt virus (RRSV) and other viral diseases have caused severe rice yield loss. T...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/85A01K67/033
CPCC12N9/0081C12Y114/15006C12N15/85A01K67/0339C12N2800/105A01K2217/05A01K2217/15A01K2227/706A01K2267/03
Inventor 吴顺凡曾彬高聪芬
Owner NANJING JIXING BIOTECH DEV CO LTD
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