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Method for silencing bradysia odoriphaga cytochrome P450 enzyme system gene, and application thereof

A technology of leek, cytochrome, which is applied in the field of molecular biology, can solve the problem of lack of cytochrome P450 enzyme gene silencing, etc., and achieve the effects of improving sensitivity, reducing drug resistance and simple operation.

Active Publication Date: 2021-08-27
JIANGSU XUHUAI DISTRICT HUAIYIN AGRI SCI RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of a fast and efficient method for gene silencing of cytochrome P450 enzymes of T.

Method used

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  • Method for silencing bradysia odoriphaga cytochrome P450 enzyme system gene, and application thereof
  • Method for silencing bradysia odoriphaga cytochrome P450 enzyme system gene, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Silence of P450 gene CYP9J35-like in T. tardiformis by RNA interference technology

[0044] 1. Test materials and sources

[0045] The indoor sensitive strain of Chive tardiformis was collected from the leek field in Shouguang City, Shandong Province, and was continuously raised for more than 80 generations without exposure to insecticides;

[0046] Trizol Reagent Total RNA Extraction Kit was purchased from Ambion, USA;

[0047] rTaq polymerase, LATaq polymerase, DNase I, PrimeScript RT-reagent Kit reverse transcription kit, pMD18-T vector and fluorescence quantitative PCR kit were all purchased from Bao Biological Engineering Co., Ltd., dsRNA synthesis kit was purchased from Thermo Fisher Scientific, primers were synthesized by BGI.

[0048] A refrigerated centrifuge was purchased from Eppendorf, Germany;

[0049] Nanodrop, purchased from U.S. Termo Scientific company;

[0050] Agilent 2100 biological analysis instrument, purchased from U.S. Agilent Technologies In...

Embodiment 2

[0078] Silence of P450 gene CYP6FV2-like in T. tardiformis by RNA interference technology

[0079] Test materials and sources are the same as in Example 1.

[0080] The test procedure is the same as that in Example 1, except that the dsRNA fed is the dsRNA of the CYP6FV2-like gene.

[0081] Wherein, the nucleotide sequence of CYP6FV2-like is shown in SEQ ID NO.2; the nucleotide sequence of the forward primer of CYP6FV2-like is shown in SEQ ID NO.6, specifically: taatacgactcactatagggATTTTTCATTGCCGGTTTTG; the reverse primer of CYP6FV2-like The nucleotide sequence of the primer is shown in SEQ ID NO.7, specifically: taatacgactcactatagggCAAGGCCAATTTTCGTTTGT.

[0082] The qRT-PCR method was used to detect the silencing efficiency of CYP6FV2-like of the silenced P450 enzyme gene CYP6FV2-like of T. chileensis. The nucleotide sequence of the CYP6FV2-like forward primer used is shown in SEQ ID NO.12, specifically: TCACCCACATTTACATCC; the nucleotide sequence of the CYP6FV2-like revers...

Embodiment 3

[0085] Silence of P450 gene CYP3A56-like in T. tardiformis by RNA interference technology

[0086] Test materials and sources are the same as in Example 1.

[0087] The test procedure is the same as that in Example 1, except that the dsRNA fed is the dsRNA of the CYP3A56-like gene.

[0088] Wherein, the nucleotide sequence of CYP3A56-like is shown in SEQ ID NO.3; the nucleotide sequence of the forward primer of CYP3A56-like is shown in SEQ ID NO.8, specifically: taatacgactcactatagggCTATGACGTCGCGAAAAACA; the reverse primer of CYP3A56-like The nucleotide sequence of the primer is shown in SEQ ID NO.9, specifically: taatacgactcactatagggTTCGGTATAACAACGCCCTC.

[0089] The qRT-PCR method was used to detect the silencing efficiency of CYP3A56-like of the silenced P450 enzyme gene CYP3A56-like of T. chileensis. The nucleotide sequence of the CYP3A56-like forward primer used is shown in SEQ ID NO.14, specifically: The nucleotide sequence of the reverse primer of GTTGGTGCTGTGCCTGTT; C...

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Abstract

The invention provides a method for silencing a bradysia odoriphaga cytochrome P450 enzyme system gene, and application thereof, and belongs to the technical field of molecular biology. The bradysia odoriphaga P450 enzyme system gene CYP9J35-like and / or CYP6FV2-like and / or CYP3A56-like is silenced by applying an RNA interference technology, the method is accurate and reliable, the operation is simple, the silencing efficiency is high, and a research basis is provided for bradysia odoriphaga drug resistance mechanism research and resistance treatment.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a gene silencing method and application of tardyophthora tardigrades cytochrome P450 enzyme system. Background technique [0002] The larvae of Chive tardiformis, commonly known as chive maggots, are important pests of Liliaceae vegetables such as onions and garlic, and they especially like to eat leeks. Improper prevention and control measures of Chive tardigrades can easily lead to reduced production of leeks or excessive pesticide residues. With the rapid development of global warming and facility agriculture, the damage of leek maggots has become more and more serious, which has seriously restricted the development of leek planting industry. [0003] At present, the prevention and control measures of tardigrades on chives are mainly chemical control, and chemical agents such as chlorpyrifos, phoxim, lambda-cyhalothrin, and thiamethoxam are applied with ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/53A01K67/033
CPCC12N15/1137A01K67/0339C12N9/0081C12Y114/15006
Inventor 陈澄宇赵云霞刘莹曹凯歌张凯
Owner JIANGSU XUHUAI DISTRICT HUAIYIN AGRI SCI RES INST
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