Cotton bollworm e75 gene and its application in RNA-mediated pest control

A cotton bollworm and gene technology, applied in the direction of DNA/RNA fragments, application, genetic engineering, etc., can solve the problems of increased resistance and aggravated damage of transgenic Bt cotton, achieving high prospective, low risk of resistance, and reduced The effect of pupation rate

Active Publication Date: 2021-11-16
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The widespread application of transgenic Bt (Bacillusthuringiensist) cotton effectively controls the harm of cotton bollworm, but the resistance of cotton bollworm to Bt cotton is also increasing significantly, and at the same time, the harm of cotton bollworm to other non-transgenic crops is also increasing year by year

Method used

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  • Cotton bollworm e75 gene and its application in RNA-mediated pest control
  • Cotton bollworm e75 gene and its application in RNA-mediated pest control
  • Cotton bollworm e75 gene and its application in RNA-mediated pest control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Obtaining the cDNA sequence of the cotton bollworm E75 gene

[0044] 1. Extraction of total RNA of cotton bollworm

[0045] The whole process of total RNA extraction of cotton bollworm is carried out in a sterile operating table, and the reagents and consumables used are RNAase-free. The specific steps are as follows:

[0046] 1) Collect fresh cotton bollworm tissue, then add it to a sterile tissue grinder frozen with liquid nitrogen, quickly add 1 ml of Trizol reagent, fully grind the tissue, and then use an RNase-free pipette tip to transfer the homogenate to a sterile tissue grinder. After centrifugation in a 1.5ml centrifuge tube of RNAase, let stand in an ice bath for 5-10min; the speed of the centrifuge tube is 12000rpm, the temperature is 4°C, and the centrifugation time is 10min;

[0047] 2) Transfer the centrifuged supernatant (about 900 μl) in step 3) to a new RNase-free centrifuge tube;

[0048] 3) Add 200 μl of chloroform to the supernatant of th...

Embodiment 2

[0055] Example 2 Cloning of E75 gene target fragment of Helicoverpa armigera and preparation of dsRNA

[0056] I. Design specific primers

[0057] According to the characteristics of the E75 gene, the gene fragments in the DNA binding region of the gene were selected, and their effects on insects were studied by injection method. The DNA binding region is an important region that regulates the transcription of insect hormone nuclear receptors and plays an important role in the regulation of insect hormones. , so this target sequence was selected to observe the effect of the gene silencing on the development and metamorphosis of cotton bollworm; the selected control gene was the green fluorescent protein GFP gene. Since the GFP gene does not exist in insects, a large number of studies have used it as a control. Gene synthesis of dsRNA;

[0058] Use Primer Premier 5.0 software to design specific primers with reference to the design principles of RNA primers. The designed specif...

Embodiment 3

[0068] Example 3 Verification that E75-dsRNA of Helicoverpa armigera inhibits the metamorphosis of Helicoverpa armigera

[0069] 1. Microinjection of dsRNA of the E75 gene of Helicoverpa armigera

[0070] Helicobacter bollworm larvae of 5 instars and 1d, of uniform size and the same health status were selected, and the control group injected with dsGFP and the experimental group injected with dsE75 were set up. The abdominal intersegmental membrane between the 8th and 9th sections was injected into the body cavity of the cotton bollworm larvae. The injection volume of dsRNA was 10 μg / head, and each group was injected with 90 heads, and 3 biological replicates were set. Cotton bollworm larvae were raised in 12-well plates with artificial feed;

[0071] 2. Detection of gene silencing effect of cotton bollworm E75

[0072] Within 0-96 hours after dsRNA injection, samples were taken every 24 hours for gene silencing effect detection. Four heads were taken from the control group ...

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Abstract

Cotton bollworm E75 gene, select the target fragment containing SEQ ID NO: 3, and then synthesize dsRNA, and then introduce the synthesized dsRNA into the 5th instar larva of cotton bollworm by microinjection, and express after the synthetic dsRNA is injected into the E75 gene of cotton bollworm Inhibited, the molting metamorphosis process of the cotton bollworm is destroyed, effectively reducing the pupation rate of the cotton bollworm, and then controlling its population; the research foundation is good, the prospect is high, and the risk of pest resistance is low, which is an important step for the creation of new biological insecticides. Provide a theoretical basis for pesticides and plant insect-resistant genetic engineering.

Description

technical field [0001] The invention relates to the field of insect growth and reproduction regulation and biotechnology, in particular to a cotton bollworm E75 gene and its application in RNA-mediated pest control. Background technique [0002] Cotton bollworm Helicoverpa armigera (Hübner) is one of the most important agricultural pests with worldwide distribution and polyphagia. It harms cotton, corn, wheat, tomato and other crops, causing huge economic losses. The widespread application of transgenic Bt (Bacillusthuringiensist) cotton has effectively controlled the harm of cotton bollworm, but the resistance of cotton bollworm to Bt cotton is also increasing significantly, and at the same time, the harm of cotton bollworm to other non-transgenic crops is also increasing year by year. Therefore, with the continuous development of society, there is an urgent need for new pest control methods to combine or replace Bt crops to achieve sustainable development of modern agricul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/113C12N15/10A01N57/16A01P7/04
CPCA01N57/16C07K14/43563C12N15/113C12N2310/14C12N2330/30
Inventor 张万娜肖海军吴珍平黄孝龙江婷许洋
Owner JIANGXI AGRICULTURAL UNIVERSITY
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