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Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control

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

Inactive Publication Date: 2015-04-29
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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  • Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control
  • Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control
  • Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 : Gypsy moth heat shock protein Hsp23 Gene full-length cloning

[0021] The gypsy moth heat shock protein Hsp23 gene has a nucleic acid sequence of 750 bp, an open reading frame of 600 bp, encoding 199 amino acids, a molecular weight of 22.84 kDa, 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 6 mers (100 μM) 0.5 μL, Total RNA 0.5 μg RNase Free 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, an...

Embodiment 2

[0024] Example 2 : Gypsy moth Hsp23 Gene dsRNA Synthesis

[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 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 20 bp T7 promoter sequence is added to the 5' end of each specific primer, and GFP is used as a control group. The target band was amplified by PCR, and the reaction program was: 94°C for 3 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 7 min, and the amplified product was confirmed by electrophoresis. Synthesize dsRNA as a template (refer to the MEGAscript RNAi Kit kit instructions), detect the concentration of dsRNA with a UV spectrophotometer, and take 0.5 µL of...

Embodiment 3

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

[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 lively 3rd instar larvae were selected at 6, 24, 48, 96, and 144 h respectively, and the RNeasy Mini animal was used to Tissue Total 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 than that of the contro...

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Abstract

The invention discloses a lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control. A nucleotide sequence and a coded amino acid sequence of the Hsp23 gene are shown by SEQ ID No.1, the dsRNA is synthesized from part of sequence fragments of the gene, and the sequence of the dsRNA is shown by SEQ ID No.2. The dsRNA of the Hsp23 gene can be applied to control of the growth and development of lymantria dispar linnaeus. An experimental result shows that after the dsRNA of the Hsp23 gene is injected into a lymantria dispar linnaeus larva, compared with a contrast (injected with ddH2O and GFP gene dsRNA), a relatively strong silencing effect is achieved, so that the growth and development of the lymantria dispar linnaeus larva are inhibited. Therefore, the invention provides a silencing technology capable of using Hsp23 to control an important forest pest, namely the lymantria dispar linnaeus and lepidopterous pests with similar structural domains, thereby providing a new train of thought and a new 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] Gypsy moth ( Lymantria dispar Linnaeus) is a worldwide pest with 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, sylvestris 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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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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