Application of heat shock protein hsp23 gene and dsrna of gypsy moth in pollution-free control

A technology of heat shock protein and gypsy moth, applied in DNA/RNA fragments, applications, genetic engineering, etc., to achieve broad application prospects, weaken vitality, and strong silencing effects

Inactive Publication Date: 2017-09-15
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the prevention and control of the important forest pest gypsy moth by inhibiting the transcription level of the small molecule heat shock protein gene

Method used

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  • Application of heat shock protein hsp23 gene and dsrna of gypsy moth in pollution-free control
  • Application of heat shock protein hsp23 gene and dsrna of gypsy moth in pollution-free control
  • Application of heat shock protein hsp23 gene and dsrna of gypsy moth in pollution-free control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Full-length cloning of the heat shock protein Hsp23 gene of the gypsy moth

[0021] The gypsy moth heat shock protein Hsp23 gene has a nucleic acid sequence of 750bp, an open reading frame of 600bp, encoding 199 amino acids, a molecular weight of 22.84kDa, and a theoretical isoelectric point PI of 5.50. It is an acidic protein.

[0022] Total RNA was extracted from Gypsy 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'-CAGTAAACAGTATTCGAAG- 3'; reverse primer: 5'-AGGCGAAAGAAACAAGCACT-3'.

[0023] Reaction system: 5×PrimeScript buffer 2 μL, PrimeScript RT Enzyme Mix I 0.5 μL, Oligo d(T) primer (50 μM) 0.5 μL, Random 6mers (100 μM) 0.5 μL, Total RNA 0.5 μg RNaseFree ddH 2 O to make up 10 μL. , PCR amplification program is as follows: 94°C for 3min; 35 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1m...

Embodiment 2

[0024] Embodiment 2: Synthesis of gypsy moth Hsp23 gene dsRNA

[0025] According to the full length of the gypsy moth Hsp23 gene cloned in Example 1, design and synthesize the Hsp23 gene dsRNA forward primer (5'-ATGTCGTTAATTCCTTACATGTA-3') and reverse primer (5'-CTAATTAGATTCTTCAATTGTCG-3'), and amplify the obtained fragment The length of the sequence is 516bp, and the dsRNA of the Hsp23 gene is obtained through an in vitro dsRNA synthesis kit.

[0026] 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 GFP 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 instructions), detect the concentration of dsRNA with a UV sp...

Embodiment 3

[0027] Example 3: Detection of silencing effect of Gypsy moth Hsp23 gene

[0028] The dsRNA (1 μg) of the Hsp23 gene and GFP gene synthesized in Example 2 was microinjected into the 3rd instar larvae of the gypsy moth, and the active 3rd instar larvae were selected at 6, 24, 48, 96, and 144 hours respectively, and the RNeasy Mini animal tissue aggregates were used to collect the dsRNA. RNA extraction kit (Qiagen) was used 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 Hsp23 gene after injection by fluorescent quantitative RT-PCR. The expression level of Hsp23 gene in the 3rd instar larvae injected with dsRNA was as follows: figure 2 shown. The results showed that the injection of dsRNA of exogenous control gene GFP into gypsy moth larvae would affect the expression of Hsp23 gene. Compared with the control GFP gene, the effect of Hsp23 gene silencing was higher ...

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Abstract

The invention discloses the application of a gypsy moth heat shock protein Hsp23 gene and its dsRNA in pollution-free control. The nucleotide sequence and encoded amino acid sequence of the Hsp23 gene are shown in SEQ ID No. 1, and dsRNA is synthesized from a partial sequence fragment of the gene, and the sequence is shown in SEQ ID No. 2. Hsp23 gene dsRNA can be used to inhibit the growth and development of gypsy moth. The experimental results showed that when the gypsy moth larvae were injected with the Hsp23 gene dsRNA, compared with the control (injection of ddH2O and GFP gene dsRNA), there was a strong silencing effect, resulting in the inhibition of the growth and development of the gypsy moth larvae. Therefore, the present invention proposes that the silencing technology of Hsp23 can be used to control the important forest pest gypsy moth and lepidopteran pests with similar structural domains, which provides a new idea and technology for green control of forest pests.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a heat shock protein Hsp23 gene of the Asian gypsy moth and its dsRNA and the application of the gene RNAi technology in the prevention and treatment of the Asian type gypsy moth. Background technique [0002] The gypsy moth (Lymantria dispar Linnaeus) is a worldwide pest with a wide distribution and miscellaneous feeding habits. According to foreign reports, it can harm more than 300 kinds of plants, and domestic reports can harm more than 500 kinds of plants such as poplar, willow, apple, Pinus sylvestris, and larch. . The gypsy moth spreads quickly, with a large amount of reproduction and food intake, causing significant economic losses to forestry production. For example, in 1974, the gypsy moth occurred in the south of Liaoning Province, eating up many oak leaves in silkworm farms, poplar, willow, elm, Hawthorn, apple leaves, etc. are also seriously damaged. At pre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/89A01N57/16A01P7/04
Inventor 曹传旺王超孙丽丽邹传山刘鹏
Owner NORTHEAST FORESTRY UNIVERSITY
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