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Insect chitin synthetase 1 gene fragment and dsRNA and application thereof

A technology of chitin synthase and gene fragments, applied in the direction of DNA/RNA fragments, applications, recombinant DNA technology, etc., can solve the problems of increased dosage, increased cost of control, and long time for killing insects, etc.

Inactive Publication Date: 2010-09-15
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The damage caused by agricultural pests is an important factor restricting agricultural production in my country. Long-term application of chemical insecticides leads to a series of problems: 1) Insects become resistant to insecticides, the dosage of pesticides increases, and the cost of control increases; 2) Pesticide residues lead to serious environmental pollution; 3) have a greater impact on non-target organisms
Existing biopesticides are widely used in pest control, but the killing time is long and the effect is slow

Method used

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  • Insect chitin synthetase 1 gene fragment and dsRNA and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0011] Example 1: Acquisition of Chitin Synthase 1 Gene Fragment 1 from Oryza chinensis

[0012] 1) Design of PCR primers

[0013] According to the amino acid conserved sequence of insect chitin synthase, the following degenerate primers were designed: upstream primer: CHS1F 5′-GAYGGNGAYATHGAYTT-3, downstream primer: CHS1R 5′-TCYTCNCCYTGRTCRTA-3′; all primers were provided by Shanghai Synthesized by Yingwei Jieji Biological Co., Ltd.

[0014] 2) Obtaining the total RNA of Oryza chinensis

[0015] Select the body surface of 5th instar nymphs of Oryza sinensis of equal size, male and female, in groups of four, and freeze them in liquid nitrogen. RNA is to be extracted. For the specific operation steps of RNA extraction, refer to the TaKaRa Trizol kit.

[0016] 3) First-strand cDNA synthesis of Oryza chinensis

[0017] First-strand cDNA synthesis steps refer to SMART TM RACE cDNA Amplification Kit.

[0018] 4) PCR amplification

[0019] Using the above-mentioned first-stra...

Embodiment 2

[0022] Example 2: Acquisition of Chitin Synthase 1 Gene Fragment 2 from Oryza chinensis

[0023] According to the nucleotide sequence of O. chinensis chitin synthase 1 gene fragment 1 obtained in Example 1, specific primers were designed using primerpremier5.0 software. The upstream primer sequence is SEQ ID NO: 3, and the downstream primer sequence is SEQ ID NO: 3. IDNO: 4, all primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd. Select the 5th instar nymphs of O. sinensis with equal size, half male and half female, in groups of four, and freeze them in liquid nitrogen for RNA extraction. For the specific operation steps, refer to the TaKaRa Trizol kit. M-MLV reverse transcriptase reverse-transcribes the extracted RNA into the first-strand cDNA, and uses this as a template to amplify the chitin synthase gene fragment by PCR. SV Gel and PCR Clean-Up System (Promega) kit for purification.

Embodiment 3

[0024] Example 3: Obtaining dsRNA of O. chinensis chitin synthase 1 gene fragment 2

[0025] Using the chitin synthase 1 gene fragment 2 obtained in Example 2, according to T7RiboMAX TM Express RNAi System (Promega) kit instructions, in vitro transcription and synthesis of dsRNA. The obtained dsRNA was detected by electrophoresis on 1.5% agarose gel, and quantified to a final concentration of 1 μg / μL by a microplate reader (Molecular Devices SpectraMax 190, Menlo Park, CA, USA). Store at -70°C for later use.

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Abstract

The invention provides an insect chitin synthetase 1 gene fragment and an application of dsRNA thereof. By cloning and measuring the sequence of Qxya chinensis chitin synthetase 1, a chitin synthetase gene 1 of which the nucleotide sequence is SEQ ID NO:1 is obtained, and a chitin synthetase gene fragment of which the sequence is SEQ ID NO:2 is selected for synthesizing the dsRNA. After the dsRNA is injected into the body cavity of an insect, the insect dies because of molting difficulty. The invention provides a new approach for developing a safe and nuisance-free insect control method.

Description

technical field [0001] The present invention relates to the field of biotechnology. It specifically relates to an insect chitin synthase 1 gene fragment and its dsRNA and the application of the dsRNA in killing pests. Background technique [0002] The damage caused by agricultural pests is an important factor restricting agricultural production in my country. Long-term application of chemical insecticides leads to a series of problems: 1) Insects become resistant to insecticides, the dosage of pesticides increases, and the cost of control increases; 2) Pesticide residues lead to serious environmental pollution; 3) Has a greater impact on non-target organisms. Existing biopesticides are widely used in pest control, but the insecticidal time is long and the effect is slow. [0003] RNA interference (RNAi) is a specific post-transcriptional gene silencing phenomenon caused by double-stranded RNA molecules, which won the Nobel Prize in 2006. The discovery of RNAi not only prov...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/113A01N61/00A01P7/04
Inventor 张建珍刘晓健马恩波杨美玲郭亚平
Owner SHANXI UNIV
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