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Hyphantria cunea tachykinin receptor gene, dsRNA and application in preventing and treating hyphantria cunea

A kind of American white moth, receptor gene technology, applied in the direction of DNA / RNA fragment, application, receptor / cell surface antigen / cell surface determinant, etc., to achieve the effect of strong silencing effect and broad application prospects

Active Publication Date: 2020-11-17
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the prevention and control of major quarantine pests American white moth by inhibiting the transcription level of insect neuropeptide receptor family genes

Method used

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  • Hyphantria cunea tachykinin receptor gene, dsRNA and application in preventing and treating hyphantria cunea
  • Hyphantria cunea tachykinin receptor gene, dsRNA and application in preventing and treating hyphantria cunea
  • Hyphantria cunea tachykinin receptor gene, dsRNA and application in preventing and treating hyphantria cunea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Full-length Cloning of the American White Moth Tachykinin Receptor Gene

[0019] The nucleotide sequence of tachykinin receptor gene of American white moth is 2379bp, the open reading frame is 1230bp, encoding 409 amino acids, the molecular weight is 47.29kDa, and the theoretical isoelectric point PI is 9.31.

[0020] Total RNA was extracted from the American white moth, using the reverse transcription kit PrimeScript TM RT reagent Kit (TaKaRa) synthesized the first strand of cDNA, and then used the first strand of cDNA as a template to design primers (forward primer: 5'-ATGAGGATGCTTGACGACCTGG- 3'; reverse primer: 5'-TCATGCAGAACTCACCAGAGTC-3').

[0021] Reaction system: 5×PrimeScript buffer 2μL, PrimeScript RT Enzyme Mix I0.5μL, Oligo d(T) primer (50μM) 0.5μL, Random 6mers (100μM) 0.5μL, Total RNA 0.5μg RNaseFree ddH 2 O to make up 10 μL. The PCR amplification program was as follows: 94°C for 3 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C fo...

Embodiment 2

[0022] Embodiment 2: Synthesis of American white moth tachykinin receptor gene dsRNA

[0023] According to the full length of the American white moth tachykinin receptor gene cloned in Example 1, design and synthesize the tachykinin receptor gene dsRNA forward primer (5'-GTACAACGTCTGTTTCATGGT-3') and reverse primer (5'-GTGCTGGTACCATTCTTCC -3'), amplified to obtain a sequence with a fragment length of 492bp, and obtaining the dsRNA of the tachykinin receptor gene through an in vitro dsRNA synthesis kit.

[0024] The specific synthesis process is that a section of T7 promoter sequence of about 20 bp is added to the 5' end of each specific primer, and EGFP is used as a control group. The target band was amplified by the PCR method, and the reaction program was: 94°C for 3min; 94°C for 30s, 60°C for 30s, 72°C for 1min, 35 cycles; 72°C for 7min. The amplified product was confirmed by electrophoresis and used as a template to synthesize dsRNA ( Refer to the MEGAscript RNAi Kit kit ...

Embodiment 3

[0025] Example 3: Detection of the silencing effect of the American white moth tachykinin receptor gene

[0026] The dsRNA (2 μg) of the tachykinin receptor gene and EGFP gene synthesized in Example 2 was microinjected into the 4th instar larvae of the American white moth, and the lively 4th instar larvae were selected at 48 and 72 hours respectively, and the total RNA of the RNeasy Mini animal tissue was used Extraction kit (Qiagen) to extract total RNA, using PrimeScript TM The first strand of cDNA was synthesized by RT kit (TaKaRa) and used as a template to detect the expression level of tachykinin receptor gene after injection by fluorescent quantitative RT-PCR. The expression level of tachykinin receptor gene in 4th instar larvae injected with dsRNA was as follows: figure 2shown. The results showed that the injection of dsRNA of the exogenous control gene EGFP into the larvae of the white moth would affect the expression of the tachykinin receptor gene. Compared with...

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Abstract

The invention discloses a hyphantria cunea tachykinin receptor gene, dsRNA and application in preventing and treating hyphantria cunea. The tachykinin receptor gene has a nucleotide sequence shown asSEQ ID No.1 and encodes a protein having an amino acid sequence shown as SEQ ID No. 2. The tachykinin receptor gene dsRNA has a nucleotide sequence shown as SEQ ID No. 3. The hyphantria cunea tachykinin receptor gene dsRNA is applied to the prevention and treatment or preparation of products for preventing and treating hyphantria cunea. The hyphantria cunea tachykinin receptor gene and dsRNA can reduce the intake of hyphantria cunea larvae, significantly reduce the hunger resistance of larvae, weaken the vitality of hyphantria cunea, and finally achieve the prevention and treatment effect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a tachykinin receptor gene (Tachykinin receptor) and dsRNA of the white moth and its application in preventing and treating the white moth. Background technique [0002] The American white moth Hyphantria cunea (Drury) belongs to the family Arctiidae of the order Lepidoptera and is native to North America (Morris, 1963). It was introduced into my country from Dandong City, Liaoning Province in 1979, and its hosts include 175 species of plants in 49 families, 108 genera (Yang Zhongqi and Zhang Yongan, 2007). Because the insect spreads quickly, has strong fecundity, damages many host plants, and consumes a large amount of food, it often breaks out and becomes a worldwide quarantine pest. At present, chemical pesticides such as diflubenzuron III (Dong Wei, 2000), 3.6% nicotine-matrine microcapsule suspension (Zhang Huawei et al., 2013) and triflubenzuron (Qi L...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113C07K14/705A01N57/16A01P7/04
CPCC07K14/70571C12N15/1138A01N57/16C12N2310/14
Inventor 孙丽丽曹传旺王志英张晨书彭萌萌闫丽琼
Owner NORTHEAST FORESTRY UNIVERSITY
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