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Beta-N-acetylglucosaminidase genes of insects and application thereof

A technology of glucosidase and acetylaminosidase, which is applied to the application field of insect β-N-acetylglucosaminidase gene sequence and dsRNA, can solve the problems of increased control cost, slow effect, toxic pesticides harming human health and the like

Inactive Publication Date: 2013-01-16
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The long-term use of organochlorine, organophosphorus and other chemical insecticides to control pests in our country has caused a series of problems: pesticide residues lead to agricultural environmental pollution; insects have resistance to insecticides, and the cost of control has increased; toxic pesticides endanger human health, etc.
At present, biopesticides have been applied to pest control, but the action time is often long and the effect is slow

Method used

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  • Beta-N-acetylglucosaminidase genes of insects and application thereof
  • Beta-N-acetylglucosaminidase genes of insects and application thereof
  • Beta-N-acetylglucosaminidase genes of insects and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0012] Embodiment 1: Obtaining of migratory locust β-N-acetylglucosaminidase gene fragment and dsRNA thereof

[0013] 1. Acquisition of β-N-acetylglucosaminidase gene fragment of migratory locust

[0014] 1) Search of migratory locust β-N-acetylglucosaminidase gene in migratory locust expressed sequence tag EST database

[0015] Based on the expressed sequence tag (EST) database of migratory locusts, bioinformatics methods were used to search the β-N-acetylglucosaminidase genes of migratory locusts. After sequence analysis and comparison, a total of 17 migratory locusts β-N -A fragment of the acetylglucosaminidase gene. After splicing, the obtained migratory locust β-N-acetylglucosaminidase gene (LmNAG1) sequence has a full length of 2667bp and an open reading frame of 1845bp.

[0016] 2) Design of primers for migratory locust β-N-acetylglucosaminidase gene

[0017] Based on the obtained base sequence of LmNAG1, primers were designed using primer premier5.0 software. All p...

Embodiment 2

[0031] Embodiment 2: migratory locust β-N-acetylglucosaminidase gene dsRNA kills 2 instar migratory locust

[0032] 1. Injection of migratory locust β-N-acetylglucosaminidase gene dsRNA

[0033] 1 μl (3 μg) of dsRNA of SEQ ID NO: 2 was injected with a 25 μl micro-syringe between the second and third abdominal segments of the second-day-old nymphs of the 2nd-instar migratory locust, and a total of 30 were injected, half male and half male. Inject dsGFP at the same volume concentration into the control group. The injected migratory locusts were raised in a constant temperature biochemical incubator at 28°C.

[0034] 2. Observation of the phenotype of the 2-year-old migratory locust after injection of dsRNA

[0035] After the 2nd instar nymphs were injected with dsRNA, 3 days after the injection of dsGFP the control group began to molt and all successfully molted to the third instar. The average molt time was 15-20 minutes. eat. There were 30 nymphs in the treatment group inj...

Embodiment 3

[0036] Embodiment 3: Migratory locust β-N-acetylglucosaminidase gene dsRNA kills 5th instar migratory locust

[0037] 1. Injection of migratory locust β-N-acetylglucosaminidase gene dsRNA

[0038] 2 μl (6 μg) of dsRNA of SEQ ID NO: 2 was injected with a 25 μl microinjector between the second and third abdominal segments of the 2nd-day-old nymphs of the 5th instar migratory locust, and a total of 40 were injected, half male and half male. Inject dsGFP at the same volume concentration into the control group. The injected migratory locusts were raised in a constant temperature biochemical incubator at 28°C.

[0039] 2. Observation of the phenotype of the 5th instar migratory locust after injection of dsRNA

[0040] After the fifth instar nymphs were injected with dsRNA, the worms in the control group began to molt 3 days later and all successfully developed into adults on the 5th day after injection. The molting time was about 20 minutes, and the worms in the control group were...

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Abstract

The invention provides a sequence of beta-N-acetylglucosaminidase genes (NAG) of insects and an application method for double-strand ribonucleic acid (dsRNA) of the beta-N-acetylglucosaminidase genes. By the dsRNA, the specific beta-N-acetylglucosaminidase genes can be silenced, so that pests are dead. After the beta-N-acetylglucosaminidase genes of migratory locusts are cloned and sequenced, the beta-N-acetylglucosaminidase genes of which the sequence is SEQ ID NO: 1 are obtained; and fragments of the genes of which the sequence is SEQ ID NO: 2 are selected to be used for synthesizing the dsRNA. After the dsRNA of the genes is injected into body cavities of the insects, the development of the insects in the growth process is slow, and the insects cannot enter the next stadium due to incapability of smooth ecdysis, so that the insects are dead. By the beta-N-acetylglucosaminidase genes and the dsRNA, a new target is provided for RNA interference-based pest control.

Description

technical field [0001] The present invention relates to the field of biotechnology. It specifically relates to the application of insect β-N-acetylglucosaminidase gene sequence and dsRNA. Background technique [0002] The long-term use of organochlorine, organophosphorus and other chemical insecticides to control pests in my country has led to a series of problems: pesticide residues lead to agricultural environmental pollution; insects develop resistance to insecticides, and the cost of control increases; toxic pesticides endanger human health and so on. At present, biopesticides have been applied to pest control, but the action time is often long and the effect is slow. In order to protect plants more effectively, there is an urgent need to develop new pest control methods. [0003] RNA interference (RNAi), a phenomenon of specific post-transcriptional gene silencing usually caused by double-stranded RNA molecules, won the Nobel Prize in 2006. This discovery is not only...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/113A01P7/04
Inventor 李大琪容烁李涛张建珍马恩波
Owner SHANXI UNIV
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