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A kind of cDNA of cotton bollworm molting hormone regulatory factor e78-c gene and its application

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 damage, increased resistance of trans-Bt cotton, etc., and achieve the effect of low risk, high foresight, and reduced egg production

Active Publication Date: 2021-11-09
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Transgenic Bt( Bacillus thuringiensist ) The widespread use of 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-GM crops is also increasing year by year

Method used

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  • A kind of cDNA of cotton bollworm molting hormone regulatory factor e78-c gene and its application
  • A kind of cDNA of cotton bollworm molting hormone regulatory factor e78-c gene and its application
  • A kind of cDNA of cotton bollworm molting hormone regulatory factor e78-c gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Obtaining the cDNA sequence of the cotton bollworm E78-C gene

[0071] 1. Extract total RNA from cotton bollworm

[0072] Insect total RNA using TRIzol ® The RNA separation and extraction reagent (TIANGEN company) was carried out under RNase-free conditions, and the specific extraction steps were as follows:

[0073] (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 use;

[0074] (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 Trizol to the glass homogenizer ® Reagent, fully ground;

[0075] (3) Transfer the ground homogenate to an RNase-free 1.5 ml centrifuge tube with an RNase-free pipette tip for centrifugation, then place it in an ice bath for 3-5 min at 4°C; the centrifuge tube rotates at 12,000 rpm , centrifugation time 15 min;...

Embodiment 2

[0102] Example 2 Preparation and Synthesis of Cotton Bollworm E78-C Gene dsRNA Template

[0103] I, preparation of cotton bollworm E78-C gene dsRNA template

[0104] (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 Dilute with nuclease water, and then use agarose gel with a concentration of 1% to detect the length of the PCR fragment;

[0105] (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, and then use 1% agarose gel And micro-ultraviolet spectrophotometer to measure the singleness and concentration of the purified product;

[0106] II. Synthesis of dsRNA

[0107] Using T7 RiboMAX TM The Express RNAi System kit synthesizes the recovered DNA by in vit...

Embodiment 3

[0128] Example 3 Screening dsRNA with E78-C gene fragments to inhibit ovarian development and oviposition in cotton bollworm

[0129] 1. Microinjection of E78-C gene dsRNA of cotton bollworm

[0130] 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 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 absorbent...

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Abstract

A kind of E78-C gene cDNA of cotton bollworm ecdysone regulatory factor, clones the ecdysone-inducible factor E78-C gene from cotton bollworm, and designs primers for the ecdysone-induced factor E78-C gene of cotton bollworm, selects SEQ ID NO. The 658-1122 position in 1 was used as the E78-C target gene fragment to synthesize dsRNA, and then the synthesized dsRNA was delivered to the body cavity of cotton bollworm by microinjection, and the green fluorescent protein (GFP) was used as the control group, and the dsRNA after injection was counted. The amount of eggs laid by cotton bollworm shows that the E78-C gene of cotton bollworm is used as the target gene for interference, and the injection interference is performed on the cotton bollworm to silence the transcription and expression of the gene, thereby inhibiting the development of the ovary of the cotton bollworm, reducing the amount of eggs laid, and then controlling its population; and The risk of pest resistance is low, which provides a theoretical basis for the creation of new biopesticides 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 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 agricultural pest with worldwide distribution and polyphagia, which seriously harms cotton, corn, wheat, tomato and other crops, causing huge economic losses. Transgenic Bt( Bacillus thuringiensist ) The widespread use of 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 mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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