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Molting hormone regulatory factor E78-C gene cDNA of cotton bollworm and application thereof

A technology of E78-C and ecdysone, which is applied in the field of insect growth and reproduction regulation and biology, can solve the problems of increased resistance and aggravated damage of cotton bollworm to Bt cotton, and achieves high prospective, low risk, and reduced oviposition volume effect

Active Publication Date: 2018-12-07
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 (Bacillus thuringiensist) 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. Intensified year by year

Method used

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  • Molting hormone regulatory factor E78-C gene cDNA of cotton bollworm and application thereof
  • Molting hormone regulatory factor E78-C gene cDNA of cotton bollworm and application thereof
  • Molting hormone regulatory factor E78-C gene cDNA of cotton bollworm and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining the cDNA sequence of the cotton bollworm E78-C gene

[0040] 1. Extract total RNA from cotton bollworm

[0041] Insect Total RNA Utilization The RNA separation and extraction reagent (TIANGEN company) was carried out under RNase-free conditions, and the specific extraction steps were as follows:

[0042] (1) Wash and dry the glass homogenizer, wrap it tightly with tin foil, put it into a high-temperature dry heat sterilizer for sterilization, take it out and cool it to room temperature for later use;

[0043] (2) Take 50-100 mg of fresh cotton bollworm tissue, add it to a glass homogenizer pre-cooled with liquid nitrogen, and quickly add 1 ml to the glass homogenizer Reagent, fully ground;

[0044] (3) Transfer the ground homogenate to an RNase-free 1.5ml centrifuge tube with an RNase-free pipette tip for centrifugation, then let it stand in an ice bath for 3 to 5 minutes at a temperature of 4°C; Time 15min;

[0045] (4) Transfer the supernat...

Embodiment 2

[0071] Preparation and synthesis of embodiment 2 Cotton bollworm E78-C gene dsRNA template

[0072] I, preparation of cotton bollworm E78-C gene dsRNA template

[0073] (i) Perform PCR amplification using the plasmid with the target gene fragment as a template. The PCR reaction system is 50 μl, and the reaction conditions are consistent with the above PCR amplification. Take 1 μl of the amplified PCR product and add 4 μl of nuclease-free water Dilution is performed, and then the length of the PCR fragment is detected using an agarose gel with a concentration of 1%;

[0074] (ii) After purifying the PCR product in step (i) using a DNA product purification kit, take 1 μl of the purified product and add 4 μl of RNase-free water to dilute it, and then use agarose gel with a concentration of 1% and a micropipette UV spectrophotometer was used to measure the purity and concentration of the purified product;

[0075] II. Synthesis of dsRNA

[0076] Using T7RiboMAX TM The Express ...

Embodiment 3

[0097] Example 3 Screening dsRNA with E78-C gene fragments to suppress ovarian development and oviposition in cotton bollworm

[0098] 1. Microinjection of E78-C gene dsRNA of cotton bollworm

[0099] Select female cotton bollworms of uniform size and health status that have been eclosion for 1 day, and set up a dsRNA control group injected with GFP and a dsRNA experimental group injected with E78-C. Before injection, female cotton bollworms were treated with CO 2 Anesthetize, and then use a micro-injector to inject dsRNA synthesized in vitro into the body cavity of cotton bollworm along the abdominal intersegmental membrane of the penultimate segment. The injection volume of dsRNA is 2 μl, the injection concentration is 5 μg / μl, and 90 heads are injected in each group. 3 biological repetitions; after the injection, the female worms of the cotton bollworm were raised in circular plastic boxes with a diameter of 8 cm and a height of 10 cm. Each plastic box was covered with abso...

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Abstract

The invention provides a molting hormone regulatory factor E78-C gene cDNA of cotton bollworm. A molting hormone induction factor E78-C gene is cloned from the cotton bollworm; a primer is designed for the molting hormone induction factor E78-C gene of the cotton bollworm; the 658th to the 1122nd in the SEQ ID No. 1 are used as an E78-C target gene fragment to synthesize dsRNA, and then the synthesized dsRNA is transferred to the body cavity of the cotton bollworm by microinjection; a green fluorescent protein (GFP) is used as a control group; and the counting of the egg laying amount of the cotton bollworm injected with dsRNA indicates that when the E78-C gene of the cotton bollworm is used as an interference target gene to carry out injection interference on the cotton bollworm and transcriptional expression of a silent scene, the ovary development of the cotton bollworm is inhibited, the egg laying amount is reduced, and further the population quantity of the cotton bollworm is controlled. The risk that injurious pests have drug resistance is low. A theoretical basis is provided for the creation of novel biological insecticides and plant pest-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 gene cDNA of E78-C gene regulating factor of cotton bollworm ecdysone and its application. Background technique [0002] Cotton bollworm Helicoverpa armigera (Hübner) is an important polyphagous agricultural pest with worldwide distribution. It seriously damages cotton, corn, wheat, tomato and other crops, causing huge economic losses. The widespread application of transgenic Bt (Bacillus thuringiensist) 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. intensified 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 dev...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10A01N37/46A01P7/04
CPCA01N37/46C07K14/43563
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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