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E93 gene and application of dsRNA of E93 gene in pest control

A technology of genes and gene fragments, applied in the field of application of migratory locust E93 gene and its dsRNA in pest control, can solve problems such as insect drug resistance, ecological environment pollution, and human health threats

Active Publication Date: 2019-03-22
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in locust control work, chemical insecticides are still used as the main method. The extensive use of chemical insecticides not only easily leads to insect resistance, but also brings serious pollution to the ecological environment and poses a threat to human health. Therefore, the development of new green pesticides is of great significance to the control of locusts in my country

Method used

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  • E93 gene and application of dsRNA of E93 gene in pest control
  • E93 gene and application of dsRNA of E93 gene in pest control

Examples

Experimental program
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Effect test

Embodiment 1

[0010] Example 1: Acquisition of full-length cDNA and gene fragments of migratory locust E93 gene

[0011] Based on the transcriptome database of migratory locust, the E93 gene of migratory locust was searched by bioinformatics method, and the E93 gene of migratory locust was obtained ( LmE93 ) full-length cDNA sequence, using primer premier 5.0 software to design upstream and downstream primers to verify the full-length cDNA sequence, and sent to Shanghai Yingwei Jieji Biological Co., Ltd. for synthesis. Three 5th-instar migratory locust nymphs with equal size and equal size were selected, and their body walls were quickly dissected under a stereomicroscope, and frozen in liquid nitrogen. RNA was extracted according to the TaKaRaRNAiso Plus kit. Use the instructions of Reverse Transcriptase M-MLV (RNase H-) to reverse-transcribe the extracted RNA into the first-strand cDNA, use this as a template, combine with the designed upstream and downstream primers, and amplify by PCR...

Embodiment 2

[0012] Example 2: Migratory locust E93 gene-specific dsRNA synthesis

[0013] 1) Design of dsRNA primers for migratory locust E93 gene

[0014] Based on the full-length cDNA sequence of migratory locust E93 gene, primer premier 5.0 software was used to design dsRNA primers, and their sequences were SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Both upstream and downstream primers carry T7 promoter sequence. All primers were synthesized by Shanghai Yingwei Jieji Biological Co., Ltd.

[0015] 2) Synthesis of dsRNA of migratory locust E93 gene

[0016] The E93 gene extraction plasmid with the above sequence of SEQ ID NO: 1 was used as a template, and SEQ ID NO: 3 and SEQ ID NO: 4 were used as upstream and downstream primers for PCR amplification. The amplified PCR product, whose sequence is SEQ ID NO: 2, was purified by FastPure GelDNA Extraction Mini Kit (Vazyme) kit, followed by T7 RiboMAX™ Express RNAiSystem (Promega) kit instructions for in vitro transcription and synthesis...

Embodiment 3

[0017] Embodiment Three: Migratory Locust E93 Gene dsRNA Lethal Migratory Locust Experiment

[0018] 1. Injection of migratory locust E93 gene dsRNA

[0019] A total of 48 5-year-old and 2-day-old nymphs with healthy growth, uniform size, and half male and half male were selected for the experimental group ds LmE93 Injection of genes for observation of phenotypes. ds that will be synthesized LmE93 Dispense 5 µL (10 µg ds LmE93 ) lightly injected between the second and third abdominal segments of the nymph's flank. At the same time, the same volume of water was injected into the control group (48 heads, half male and half male). In addition, a total of 9 5-instar 2-day-old nymphs with healthy growth and the same size were selected for the experimental group ds LmE93 Gene injection is used to test the gene silencing efficiency, and the same volume of water is injected into the control group (9 heads) at the same time. The injected migratory locusts were reared in an ar...

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Abstract

The invention provides a full-length cDNA sequence of insect E93 gene and the application of dsRNA of the E93 gene in pest control. Specifically, the full-length cDNA sequence of the E93 gene having asequence of SEQ ID NO: 1 is acquired from a locusta migratoria transcriptome database by a bioinformatics method; the dsRNA of the gene is designed and synthesized according to SEQ ID NO: 1, and canbe injected into locusta migratoria coelom to specifically silence a target gene, so that the growth and the development of locusta migratoria are hindered to cause death; and multiple experiments show that the mortality rate reaches 100%. Since specificity and high mortality rate of the dsRNA of the E93 gene provided by the invention have an important practical significance on pest control, a novel way can be provided for pest control.

Description

technical field [0001] The invention relates to the fields of biotechnology and agricultural pest control. It specifically relates to the application of migratory locust E93 gene and dsRNA in pest control. Background technique [0002] migratory locust Locusta migratoria , is an intercontinental agricultural pest, mainly distributed in Asia, Europe, Africa, and Australia. It has the characteristics of explosiveness, clustering and migration. Once it occurs, it will not only involve a wide range of areas, but also be fierce and cause serious disasters. At present, in locust control work, chemical insecticides are still used as the main method. The extensive use of chemical insecticides not only easily leads to insect resistance, but also brings serious pollution to the ecological environment and poses a threat to human health. Therefore, the development of new green pesticides is of great significance to the control of locusts in my country. [0003] RNA interference (RNAi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113A01N57/16A01P7/04
CPCA01N57/16C07K14/43563C12N15/113C12N2310/14
Inventor 刘晓健郭俊张学尧张建珍马恩波
Owner SHANXI UNIV
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