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Gene silencing technology-based gypsymoth chitin deacetylase gene

A technology of deacetylase and chitin, applied in the field of agricultural biology, can solve environmental pollution and other problems, achieve the effects of reducing production costs, increasing economic benefits, and protecting the environment

Inactive Publication Date: 2016-04-06
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. It is necessary to continuously increase the use of pesticides to achieve satisfactory control effects, resulting in more serious environmental pollution and forming a vicious circle.

Method used

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  • Gene silencing technology-based gypsymoth chitin deacetylase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Chitin deacetylase gene fragment from Gypsy moth Ld Acquisition of Cda2

[0014] According to the gypsy moth chitin deacetylase gene obtained from the gypsy moth transcriptome database Ld For the nucleotide sequence of Cda1, specific primers were designed using primerpremier5.0 software. The sequence of the upstream primer is SEQ ID NO.3, and the sequence of the downstream primer is SEQ ID NO.4. All primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd. Gypsy moth chitin deacetylase gene Ld The recombinant pEASY-T1 plasmid of Cda1 was used as a template, and the chitinase gene fragment of Gypsy moth was amplified by PCR Ld Cda2, purified using the MicroEluteCyclePureKit (OMEGA) kit.

Embodiment 2

[0015] Example 2: Chitin deacetylase gene fragment from Gypsy moth Ld Obtaining dsRNA of Cda2

[0016] Using the chitin deacetylase gene fragment obtained in Example 1 Ld Cda2, according to the instructions of T7RiboMAX? ExpressRNAiSystem (Promega) kit, in vitro transcription and synthesis of dsRNA. The obtained dsRNA was detected by 1.5% agarose gel electrophoresis, its concentration was detected by a microplate reader (ThermoScientific MultiskanGO, ThermoFisher Scientific, USA), and concentrated to a final concentration of 1 μg / μl and stored at -80°C for later use.

Embodiment 3

[0017] Example 3: Chitin deacetylase gene fragment from Gypsy moth Ld Gypsy moth assay of dsRNA of Cda2

[0018] 1) Chitin deacetylase gene fragment of Gypsy moth Ld Cda2 dsRNA injection

[0019] Pupal stage larvae were selected for injection of the above-mentioned synthesized dsRNA. A 5ul micro-syringe is used for injection. Do not use too much force when injecting. The flank is used as the injection point, and the valve should be avoided. The amount of dsRNA injected was 5 μg, and a control group injected with the same amount of DEPC-treated water was set up, and 20 heads in each of the experimental group and the control group. After the injection, the larvae were placed in a biochemical incubator purchased from the Institute of Forest Ecology, Environment and Protection of the Chinese Academy of Forestry for breeding (light:dark time=16h:8h, temperature 26±1°C, humidity 70%).

[0020] 2) Observation of the phenotype of the gypsy moth after dsRNA injection

[0021] Af...

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Abstract

The invention relates to the technical field of agricultural biology, in particular to a gene silencing technology-based gypsymoth chitin deacetylase gene, a gene segment of the gene silencing technology-based gypsymoth chitin deacetylase gene, dsRNA of the gene segment and application of the dsRNA in affecting a pest development process. According to the gene silencing technology-based gypsymoth chitin deacetylase gene provided by the invention, gene segment LdCda2 and the dsRNA of the gene segment LdCda2 are obtained according to gypsymoth chitin deacetylase gene LdCda1, and by using the dsRNA, the development of insects is retarded due to the fact that the insects are impaired in a metamorphosis process, the large using amount of pesticides can be hopefully reduced, the environment is protected, the production cost is reduced, the economic benefit is improved, the gene silencing technology-based gypsymoth chitin deacetylase gene has a market application prospect, and a new approach is provided for the development of a safe and nuisanceless pest control method.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a gypsy moth chitin deacetylase gene and its gene fragment based on gene silencing technology, and the application of dsRNA and dsRNA of the gene fragment in the process of affecting the development of pests. 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. It is necessary to continuously increase the use of pesticides to achieve satisfactory control effects, resulting in more serious environmental pollution and forming 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 severe cases even produce rashes. Therefore, in the practice of agricultural production, there is an urgent need for alternative control methods ot...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/10C12N15/113A01N57/16A01P7/04
CPCC12N9/80A01N57/16C12N15/10C12N15/1137C12N2310/14
Inventor 范晓军赵秋勇李瑶糜艳霞杨春
Owner TAIYUAN UNIV OF TECH