Lymantria dispar linnaeus glutathione-S-transferase GSTe4 gene, dsRNA and application in prevention and treatment of lymantria dispar linnaeus

A technology of glutathione and gypsy moth, applied in the direction of transferase, DNA/RNA fragments, applications, etc., to achieve strong silencing effect, broad application prospects, and prolonged growth and development

Active Publication Date: 2020-12-11
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 about controlling the important forest pest gypsy moth by inhibiting the transcription level of GST gene family genes

Method used

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  • Lymantria dispar linnaeus glutathione-S-transferase GSTe4 gene, dsRNA and application in prevention and treatment of lymantria dispar linnaeus
  • Lymantria dispar linnaeus glutathione-S-transferase GSTe4 gene, dsRNA and application in prevention and treatment of lymantria dispar linnaeus
  • Lymantria dispar linnaeus glutathione-S-transferase GSTe4 gene, dsRNA and application in prevention and treatment of lymantria dispar linnaeus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Gypsy moth glutathione-S-transferase GSTe4 gene full-length cloning

[0022] The nucleotide sequence of the glutathione-S-transferase GSTe4 gene of the gypsy moth is 960bp, the open reading frame is 678bp, encoding 225 amino acids, the molecular weight is 25.40kDa, and the theoretical isoelectric point PI is 7.06.

[0023] 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'-ATCAGTTTATCTTTGTTTACGACT- 3'; reverse primer: 5'-AAGCACACTAATGAAACTAAAAC-3').

[0024] Reaction system: 5×PrimeScript buffer 2μL, PrimeScript RT Enzyme Mix I 0.5μL, 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 for...

Embodiment 2

[0025] Embodiment 2: Synthesis of gypsy moth GSTe4 gene dsRNA

[0026] According to the full length of the gypsy moth GSTe4 gene cloned in Example 1, design and synthesize the GSTe4 gene dsRNA forward primer (5'-GATGGAGCATAAAACA-3') and reverse primer (5'-GCGGGTGCGTTGATAGTCTTG-3'), and amplify the fragment The length of the sequence is 516bp, and the dsRNA of the GSTe4 gene is obtained through an in vitro dsRNA synthesis kit.

[0027]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 spectrophotometer, an...

Embodiment 3

[0028] Example 3: Detection of the silencing effect of the gypsy moth GSTe4 gene

[0029] The dsRNA (1 μg) of the GSTe4 gene and the 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 48, 72 and 120 hours respectively, and the RNeasy Mini animal tissue total RNA extraction kit was used (Qiagen) to extract total RNA using PrimeScript TM The RT kit (TaKaRa) synthesized the first strand of cDNA, which was used as a template, and the expression level of GSTe4 gene after injection was detected by fluorescent quantitative RT-PCR. The expression level of GSTe4 gene in the 3rd instar larva of Gypsy moth 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 GSTe4 gene. Compared with the control GFP gene, the effect of GSTe4 gene silencing was higher than that...

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Abstract

The invention discloses a lymantria dispar linnaeus glutathione-S-transferase GSTe4 gene, dsRNA and application in prevention and treatment of lymantria dispar linnaeus. The nucleotide sequence of thetransferase GSTe4 gene is as shown in SEQ ID No. 1, and the transferase GSTe4 gene encodes a protein with an amino acid sequence as shown in SEQ ID No. 2. The nucleotide sequence of the dsRNA of thetransferase GSTe4 gene is as shown in SEQ ID No. 3. The invention also discloses application of the lymantria dispar linnaeus glutathione S-transferase GSTe4 gene dsRNA in prevention and treatment orpreparation of lymantria dispar linnaeus prevention and treatment products. According to the method, physiological metabolism of the lymantria dispar linnaeus larvae to poplar secondary substances canbe affected, so that the growth and development duration of the lymantria dispar linnaeus larvae is prolonged, the weight of the larvae is reduced, the food conversion rate (ECD) is reduced, the purpose of weakening the vitality of the lymantria dispar linnaeus larvae is achieved, and finally the prevention and treatment effect is achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a glutathione-S-transferase GSTe4 gene and dsRNA of the Asian gypsy moth and its application in controlling the 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, has a large amount of reproduction and food intake, and causes significant economic losses to forestry production. At present, the control of Asian gypsy moth is still mainly based on chemical insecticides such as beta-cypermethrin and deltamethrin, but these compounds are likely to cause the formation of pest resistance and environmental pollution. Although some...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/113A01N57/16A01P7/04
CPCC12N9/1088C12Y205/01018C12N15/1137A01N57/16C12N2310/14Y02A40/146
Inventor 曹传旺孙丽丽高源张晨书庞欣茹闫丽琼马静怡王建国王振越许力山
Owner NORTHEAST FORESTRY UNIVERSITY
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