Gypsymoth chitinase 10 gene based on gene silencing technology and dsRNA

A technology of chitinase and gypsy moth, applied in DNA/RNA fragments, genetic engineering, DNA preparation, etc., can solve environmental pollution and other problems, achieve the effects of reducing production costs, reducing mass use, and protecting the environment

Inactive Publication Date: 2014-12-17
TAIYUAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In our country, the continuous single long-term use of chemical agents has caused insects to have different degrees of resistance to various pesticides. Environmental pollution, forming a vicious circle

Method used

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  • Gypsymoth chitinase 10 gene based on gene silencing technology and dsRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The method for obtaining the full length of the chitinase 10 gene of the gypsy moth is as follows:

[0016] (1) Design of PCR primers

[0017] According to the known amino acid conservation sequence of insect chitinase 10, the following degenerate primers were designed:

[0018] Upstream primer: CHT10F 5'-GACCTBGAYTGGGARTAYCC-3',

[0019] Downstream primer: CHT10R 5'-GTCTTCRAAVGAHACCCATTGRTC-3';

[0020] All primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd.

[0021] (2) Obtaining total RNA of gypsy moth

[0022] The 4th instar larvae of the gypsy moth with the same size were selected, and a group of 3 were frozen in liquid nitrogen. Total RNA was to be extracted. The specific operation steps for extracting total RNA were according to TaKaRa's Trizol kit.

[0023] (3) Synthesis of first-strand cDNA of Gypsy moth

[0024] The operating steps for the synthesis of the first strand of cDNA refer to the PrimeScriptTM RT reagent Kit with gDNA Eraser ...

Embodiment 2

[0031] The method for obtaining the lethal fragment of the gypsy moth chitinase 10 gene is as follows:

[0032] According to the nucleotide sequence of the gypsy moth chitinase 10 gene 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: 4 , all primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd.; among them, the upstream primer and downstream primer need to be designed for the nucleotide sequence of the gypsy moth chitinase 5 gene obtained in Example 1. After the design of upstream and downstream primers and careful screening of multiple gene fragments, the lethal gene fragment with practical application effect was finally determined.

[0033] The recombinant pEASY-T1 plasmid successfully ligated with the gypsy moth chitinase 10 gene obtained in Example 1 was used as a template, and the lethal fragment of the chitinase gene was o...

Embodiment 3

[0036] The method for obtaining the dsRNA of the lethal fragment of the chitinase 10 gene of the gypsy moth is as follows:

[0037] The chitinase 10 gene lethal fragment obtained in Example 2 was used to synthesize dsRNA by in vitro transcription according to the instructions of the T7RiboMAX® Express RNAi System (Promega) kit. The obtained dsRNA was detected by 1.5% agarose gel electrophoresis, and its concentration was detected by a microplate reader (Thermo Scientific Multiskan GO, Thermo Fisher Scientific, USA). Store at -80°C for later use.

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Abstract

The invention provides a genetic lethal fragment of an insect chitinase gene 10 and an application of dsRNA of the fragment. The chitinase 10 gene with the nucleotide sequence of SEQIDNO: 1 is obtained by cloning and sequencing the gypsymoth chitinase 10 gene, and the genetic fragment of chitinase with the sequence of SEQIDNO: 2 is selected for synthesizing the dsRNA. After the dsRNA is injected into a body cavity of gypsymoth, gypsymoth is dead due to hard ecdysis. The invention provides a new path for researching a safe and pollution-free insect preventing and controlling method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gypsy moth lethal gene fragment Chitinase10 and its dsRNA based on gene silencing technology. Background technique [0002] In our country, the continuous single long-term use of chemical agents has caused insects to have different degrees of resistance to various pesticides. Environmental pollution forms a vicious circle. In addition, gypsy moths can also affect human health. When people come into direct contact with gypsy moths, redness, swelling and itching often occur, and even rashes occur in severe cases. Therefore, in the practice of agricultural production, there is an urgent need for alternative control methods other than chemical pesticides. [0003] RNA interference (RNA interference, RNAi) is a gene silencing phenomenon mediated by double-stranded RNA. The double-stranded RNA is finally processed into a small RNA (siRNA) with a size of about 22 nt, which binds to the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56A01P7/04C12N15/113C12N15/10A01N61/00
Inventor 范晓军赵秋勇刘建红张常糜艳霞
Owner TAIYUAN UNIV OF TECH
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