Chitinase genes of insects and application of dsRNA thereof

A chitinase gene, insect technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve problems such as non-target biological impact, long insecticidal time, and environmental pollution

Inactive Publication Date: 2010-08-18
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 the main restricting factor of my country's agricultural production. Long-term application of chemical pesticides has caused a series of problems: 1) Insects have resistance to insecticides, the dosage of pesticides has increased, and the cost of control has increased; 2) Pesticide residues have caused serious environmental pollution; 3) have a greater impact on non-target organisms
Existing biopesticides are also widely used in pest control, but the insecticidal time is long and the effect is slow

Method used

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  • Chitinase genes of insects and application of dsRNA thereof
  • Chitinase genes of insects and application of dsRNA thereof
  • Chitinase genes of insects and application of dsRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Acquisition of Chitinase Gene Fragment and dsRNA of East Asian Migratory Locust

[0013] 1. Acquisition of Chitinase Gene Fragment of East Asian Migratory Locust

[0014] 1) Search of chitinase gene of migratory locust in migratory locust expressed sequence tag EST database

[0015] Based on the expressed sequence tag (EST) database of migratory locust, the chitinase gene of migratory locust was searched by bioinformatics method. After sequence analysis and comparison, a total of 7 chitinase gene fragments were obtained.

[0016] 2) Design of RACE primers for migratory locust chitinase gene

[0017] Based on the base sequence of the obtained chitinase gene fragment, it was designed using primer premier5.0 software. All primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd.

[0018] 3) Obtaining total RNA from migratory locust

[0019] Select the 5th instar nymphs of migratory locust with equal size, half male and half male, in groups of f...

Embodiment 2

[0042] Example 2: Chitinase gene dsRNA kills migratory locust

[0043] 1. Injection of chitinase gene dsRNA in East Asian migratory locust

[0044] Inject 2 μl (about 6 μg) of dsRNA of SEQ ID NO: 2 or SEQ ID NO: 3 with a 25 μl micro-syringe between the second and third abdominal segments of any instar nymph of migratory locust, and inject 3 biological replicates in total, 10 in each group. Only, half male and half male. Inject dsGFP at the same volume concentration into the control group. After injection, migratory locusts were raised in a constant temperature biochemical incubator at 28°C.

[0045] 2. Observation of the phenotype of the East Asian migratory locust after injection of dsRNA

[0046] There was no significant difference in food intake, body shape change and weight gain between the experimental group injected with dsRNA migratory locust and the control group. During the molting process, the migratory locusts in the control group molted smoothly, and it only took...

Embodiment 3

[0054] Embodiment 3: Chitinase gene dsRNA kills Oryza chinensis

[0055] 1. dsRNA injection of O. chinensis SEQ ID NO: 3

[0056] 2 μl (about 6 μg) of dsRNA of SEQ ID NO: 3 was injected with a 25 μl micro-syringe between the second and third abdominal segments of any instar nymphs of O. sinensis, and a total of 2 groups of biological replicates were injected. Inject dsGFP at the same volume concentration into the control group. After injection, O. sinensis was raised in a constant temperature biochemical incubator at 28°C.

[0057] 2. Observation of Oryza chinensis after injection of dsRNA

[0058] There was no significant difference between the dsRNA-injected group and the control group in food intake, body shape change and weight gain in the middle age. During the molting process, the rice locusts in the control group molted smoothly, and it only took a few minutes from the cracking of the ridge line to the complete molt of the body; while in the dsRNA group, the ridge li...

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Abstract

The invention provides chitinase gene sequences and application of dsRNA thereof, which can silence specific chitinase genes and make insects die of retarded development. By cloning, sequencing and analyzing expression of the chitinase genes of a plurality of locusta migratoria manilensis, the chitinase genes with a sequence of SEQ IDNO:1 are obtained; and the chitinase genes with sequences of SEQ ID NO:2 and SEQ ID NO:3 are selected and used for synthesizing the dsRNA. After the dsRNA is injected into a coelom of an insect, the insect shows difficult molting in the development process and dies. The chitinase gene sequences provide a basis for the effective prevention and control of the insects based on RNA interference, and provide a new approach for the development of a safe and pollution-free method for preventing and controlling the insects.

Description

technical field [0001] The present invention relates to the field of biotechnology. It specifically relates to the application of insect chitinase gene sequence and dsRNA. Background technique [0002] The damage caused by agricultural pests is the main restricting factor of my country's agricultural production. Long-term application of chemical pesticides has caused a series of problems: 1) Insects have resistance to insecticides, the dosage of pesticides has increased, and the cost of control has increased; 2) Pesticide residues have caused serious environmental pollution; 3) Has a greater impact on non-target organisms. Existing biopesticides are also widely used in pest control, but the insecticidal time is long and the effect is slow. [0003] RNA interference (RNAi), a phenomenon of specific post-transcriptional gene silencing usually caused by double-stranded RNA molecules, was awarded the Nobel Prize in 2006. This discovery not only provides a methodological breakt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/113A01N61/00A01P7/04
Inventor 张建珍李大琪张建琴马恩波郭亚平杨美玲
Owner SHANXI UNIV
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