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Application of rab5 gene and its dsRNA in migratory locust control

A technology of migratory locusts and genes, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve problems such as abnormal development, death, and abnormal life activities of insects, and achieve high lethality and insecticidal specificity Strong, highly degradable effect

Active Publication Date: 2022-07-19
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After silencing the target gene, the life activities of insects cannot be carried out normally, resulting in their abnormal development and eventually death

Method used

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  • Application of rab5 gene and its dsRNA in migratory locust control
  • Application of rab5 gene and its dsRNA in migratory locust control

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Sequence of locust small GTPase gene 5 (LmRab5) and acquisition of dsRNA

[0026] 1. Obtaining the locust small GTPase gene 5 (LmRab5) sequence

[0027] 1) Search the locust Rab5 gene sequence in the locust transcriptome database

[0028] Based on the migratory locust transcriptome database, the sequence of Rab5 gene was searched by bioinformatics method. After sequence analysis and alignment, a total of 1 sequence of Rab5 gene was obtained.

[0029] 2) Design of primers required for PCR amplification:

[0030] The upstream and downstream primers were designed according to the above-mentioned gene fragments using primer premier 5.0 software. The primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0031] 3) PCR amplification reaction

[0032] The total RNA of the third instar nymphs was extracted, and the extracted RNA was reverse transcribed into first-strand cDNA using ...

Embodiment 2

[0045] Experiment of killing migratory locusts by dsRNA of locust small GTPase gene 5 (LmRab5)

[0046] 1. Specific dsRNA Injection

[0047] 2 μl (6 μg) of the dsRNA synthesized in the above Example 1 was injected with a 10 μl specification microsyringe into the second and third abdominal segments of the second day instar nymphs of the third instar migratory locust. The control group was injected with the same volume and concentration of dsEGFP. The injected locusts were raised in a constant temperature incubation room at 30°C (light: dark time=14h:10h, temperature 30±2°C, humidity 60%), and the control and treatment groups were fed fresh wheat seedlings and wheat bran every day.

[0048] 2. Silence detection of locust small GTPase gene 5 (LmRab5)

[0049] 6 migratory locusts injected with dsEGFP (control group) and dsLmRab5 (experimental group) for 24 hours were collected, the whole nymphs were taken for total RNA extraction, and reverse-transcribed into first-strand cDNA, ...

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Abstract

The invention relates to the field of biotechnology, in particular to the application of the migratory locust Rab5 gene and its dsRNA in the control of migratory locusts. In the present invention, the fragment of the small GTPase gene 5 of the migratory locust is obtained from the transcriptome of the migratory locust specifically by the method of bioinformatics, the corresponding primers are further designed and synthesized, PCR amplification is performed, and after the amplification product is sequenced, the Rab5 gene of the migratory locust is obtained. , the sequence is shown in SEQ ID NO: 1. Further, according to SEQ ID NO: 1, design and synthesize corresponding primers, amplify by PCR to obtain a gene fragment whose nucleotide sequence is shown in SEQ ID NO: 2, and transcribe the gene fragment in vitro to synthesize dsRNA according to the kit instructions, and the After being injected into the body cavity of migratory locusts, it can specifically silence the target gene, so that the migratory locusts die when they molt. Many experiments have shown that the lethality rate reaches 100%. Due to the specificity and efficient lethality of the dsRNA of the present invention, it has important practical significance for the control of migratory locusts, and can provide a new way for the control of migratory locusts.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a locust Rab5 gene and its application in the control of migratory locusts. Background technique [0002] Due to the characteristics of migration, strong reproductive ability and mixed feeding habits, migratory locusts have a wide distribution range. Once the locust plague breaks out, it will cause serious harm to food production. At present, the prevention and control of locust plague is mainly chemical pesticides. The long-term use of chemical pesticides has brought many problems such as the generation of locust drug resistance, pesticide residues, hidden dangers of food safety and environmental pollution. Therefore, the research and development of green and effective new biological pesticides is imminent. [0003] RNA interference (RNAi) refers to the specific post-transcriptional gene silencing phenomenon that is highly conserved in evolution and triggered by double-stranded RNA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/10C12N15/113C12N15/11A01N63/60A01P7/04
Inventor 王艳丽范云鹤李涛史学凯张建珍
Owner SHANXI UNIV
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