Application of type II chitinase gene specificity dsRNA (double strand ribonucleic acid) of insect

A chitinase gene and insect technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve problems such as serious environmental pollution, rising production costs, and increasing pesticide dosages

Inactive Publication Date: 2014-09-17
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] For agricultural pests, my country has long-term use of chemical pesticides such as organochlorine and organophosphorus to control them, which has caused a series of problems: the emergence of...

Method used

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  • Application of type II chitinase gene specificity dsRNA (double strand ribonucleic acid) of insect
  • Application of type II chitinase gene specificity dsRNA (double strand ribonucleic acid) of insect
  • Application of type II chitinase gene specificity dsRNA (double strand ribonucleic acid) of insect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Obtaining dsRNA specific to migratory locust type II chitinase gene

[0014] 1. Amino acid sequence analysis of migratory locust type Ⅱ chitinase gene

[0015] Based on the transcriptome database of migratory locusts, the bioinformatics method was used to search the type Ⅱ chitinase gene of migratory locusts. The sequence encodes 2964 amino acids. Its functional domain analysis was carried out on the SMART website (http: / / smart.embl-heidelberg.de / ), and its unique chitin binding domain 1 ( figure 1 ).

[0016] 2. Gene-specific dsRNA synthesis of migratory locust type Ⅱ chitinase gene

[0017] 1) Design of dsRNA primers specific to migratory locust type Ⅱ chitinase gene

[0018] Based on the unique chitin-binding domain 1 sequence of migratory locust type II chitinase gene, it was designed using primer premier 5.0 software. Design dsRNA primers, the sequences of which are SEQ ID NO: 2 and SEQ ID NO: 3, respectively. All primers were synthesized by Shangh...

Embodiment 2

[0021] Example 2: Five-instar migratory locusts killed by migratory locust type II chitinase gene-specific dsRNA

[0022] 1. Injection of dsRNA specific for migratory locust type Ⅱ chitinase gene

[0023] 2 μl (6 μg) of dsRNA (dsCBD) of SEQ ID NO: 1 was injected with a 25 μl microsyringe between the second and third abdominal segments of the second-day-old nymphs of the fifth-instar migratory locust, and a total of 30 were injected, with half males and half females. Inject dsGFP at the same volume concentration into the control group. The injected migratory locusts were raised in a constant temperature biochemical incubator at 30°C.

[0024] 2. Gene silencing detection of migratory locust type Ⅱ chitinase

[0025] For detecting the silencing efficiency of the fifth instar dsRNA 24h after injection, the whole worm body was used as the object of RNA extraction, and 3 biological replicates were set up for each group. The total RNA of each sample was extracted and reverse-trans...

Embodiment 3

[0028] Example 3: The specific dsRNA of migratory locust type Ⅱ chitinase gene has no effect on the development of the larvae of Tribulus chinensis.

[0029] 1. Injection of the migratory locust type Ⅱ chitinase gene-specific dsRNA into Orynotus chinensis

[0030] 400ng of dsRNA (dsCBD) of SEQ ID NO: 1 was injected into the larvae of Tribulus chinensis with a microinjector, and a total of 28 larvae were injected, half male and half male. The same mass of dsGFP was injected into the control group. After the injection, the larvae of Tribulus chinensis were reared in a constant temperature biochemical incubator at 30°C.

[0031] 2. Detection of gene silencing of Chitinase type Ⅱ in Tribulus chinensis

[0032] For the detection of silencing efficiency 24 hours after the injection of dsRNA in the larvae of Trichobolus chinensis, the whole larvae were used as the object of RNA extraction, and 3 biological replicates were set up for each group. The total RNA of each sample was ext...

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Abstract

The invention provides application of a type II chitinase gene (Cht10) specificity dsRNA (double strand ribonucleic acid) of an insect. The type II chitinase gene specificity dsRNA can silence the type II chitinase gene so as to allow a migratory locust to die without affecting other insects. The type II chitinase gene of the migratory locust is cloned and sequenced, then the functional domain is analyzed, unique chitin binding domains are selected, the gene segment with the sequence of SEQ ID NO:1 is designed for synthesis of specificity dsRNA. After the dsRNA is injected into the body cavity of the migratory locust, the migratory locust cannot exuviate smoothly to step into next instar and dies at the rate of 100%, but other insects injected with the dsRNA do not die. The invention provides a target for specific pest control based on RNA interference, and the type II chitinase gene specificity dsRNA can be used for preventing and controlling specific insect-migratory locust.

Description

technical field [0001] The present invention relates to the field of biotechnology. It specifically relates to the application of insect type II chitinase gene-specific dsRNA. Background technique [0002] For agricultural pests, my country has long-term use of chemical pesticides such as organochlorine and organophosphorus to control them, which has caused a series of problems: the emergence of insecticide resistance, the need to increase the dosage of pesticides, and the increase in production costs; pesticide residues lead to serious environmental pollution and endanger human beings good health etc. With the development of society, new pest control methods are urgently needed. The invention provides a pest control application based on RNA interference. [0003] RNA interference (RNAi) is a specific post-transcriptional gene silencing phenomenon usually caused by double-stranded RNA molecules. It is one of the top ten hot technologies since the 21st century and won the N...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/113C12N15/10A01N61/00A01P7/04
Inventor 李大琪张建琴马恩波张建珍孙毅
Owner SHANXI UNIV
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