The p-glycoprotein gene of cotton bollworm and its application

A P-glycoprotein, cotton bollworm technology, applied in the field of genetic engineering and RNA interference, can solve problems such as environmental pollution, harmful genetic modification, higher animal damage, etc., and achieve the effects of solving ecological deterioration, enhancing sensitivity, and improving pest control effects.

Active Publication Date: 2019-01-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the harm of a large amount of chemical pesticides to the environment and higher animals cannot be ignored. On the one hand, it brings serious environmental pollution and becomes an important disease inducer, leading to irreversible and harmful genetic modification; These organisms are highly resistant to

Method used

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  • The p-glycoprotein gene of cotton bollworm and its application
  • The p-glycoprotein gene of cotton bollworm and its application
  • The p-glycoprotein gene of cotton bollworm and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning of the full-length sequence of the P-glycoprotein gene HaPgp1 of cotton bollworm

[0022] Extract total RNA of cotton bollworm, reverse transcribe to obtain cDNA, use cDNA as template, design primers F1 and R1 according to the 411bp sequence announced in NCBI (GenBank: KC713821.1), PCR amplification obtains the partial sequence of P-glycoprotein gene of cotton bollworm, Then use 5'-Full RACE Kit and 3'-Full RACECore Set with PrimeScript RTase (purchased from TaKaRa) by cDNA end rapid cloning technology (RACE technology), and operate according to the instructions of 5'RACE and 3'RACE kits respectively to obtain cotton bolls Insect P-glycoprotein gene cDNA sequence full-length (SEQ ID NO: 2); Then using cDNA as a template, design full-length primers F2 and R2 to amplify a band of about 3800bp size, 1% agarose gel electrophoresis analysis ( figure 1 ), and carry out cutting and recycling. Recovery fragments with pEASY TM -The Blunt zero Cloning Vector v...

Embodiment 2

[0026] Example 2 Synthesis of Cotton Bollworm HaPgp1 Gene dsRNA

[0027] According to the sequence (SEQ IDNO:3) of a length of 282bp in the HaPgp1 gene of the cotton bollworm obtained by cloning according to Example 1, the dsRNA of the HaPgp1 gene is designed, and the dsRNA is synthesized by an in vitro transcription reagent and mixed with the cotton bollworm artificial diet to feed the 2nd instar cotton boll Worm larvae.

[0028] Specifically, primers 5′-TAATACGACTCACTATAGGGGACGTCATTGATGGCAGTGT-3′ and 5′-TAATACGACTCACTATAGGGACGCTGTTTCTGACCTCCTG-3′ (SEQ ID NO: 9 and 10) were designed using the MEGAscript T7 transcription kit, and the dsRNA of the HaPgp1 gene was obtained by in vitro transcription. The dsRNA was mixed with the artificial diet of cotton bollworm (35 μg / g), fed to the 2nd instar larvae, and the expression of HaPgp1 gene was detected by fluorescence quantitative PCR at 12h, 24h, 36h and 48h after feeding, and it was found that the interference effect was the best ...

Embodiment 3

[0030] Example 3 dsRNA of HaPgp1 gene can improve the sensitivity of cotton bollworm to plant secondary substances 2-tridecanone and abamectin

[0031] The dsRNA synthesized in vitro in Example 2 is mixed with artificial feed (35 μ g / g), fed to the 2nd instar larvae of cotton bollworm, and the cotton bollworm is treated with 2-tridecanone and abamectin medicaments simultaneously, and the effect of cotton bollworm on 2 - Sensitivity to tridecanone and abamectin.

[0032]Specifically, the treatment group treated 25 2nd instar larvae of cotton bollworm with HaPgp1 dsRNA at a concentration of 35 μg / g for 12 hours, and then treated the above cotton bollworm with 0.1 mg / g 2-tridecanone and 0.4 μg / g abamectin , wherein feeding GFP dsRNA was used as a control group, DEPC water treatment was used as a negative control, and the experiment was repeated four times. The mortality of cotton bollworms in the control group and the treatment group were recorded as a standard to judge whether ...

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Abstract

The invention relates to a cotton bollworm P-glycoprotein gene and application thereof. A cotton bollworm P-glycoprotein gene HaPgp1 is obtained through cloning for the first time. The dsRNA (ribose nucleic acid) sequence designed and synthesized by using the gene HaPgp1 as a target spot on the basis of a RNA interference technology can be used for interfering the expression of the cotton bollworm P-glycoprotein gene, so that the metabolic capability of cotton bollworms on insecticides is reduced; the sensibility of insects on plant secondary substances and biopesticide is enhanced; the pest control effect of the insecticide is improved; meanwhile, positive significance is realized for solving the problems of ecological degradation caused by chemical insecticide, insecticide resistance and the like.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and RNA interference, in particular to the P-glycoprotein gene of cotton bollworm and its application. Background technique [0002] In the history of agricultural development in the world, the invention and use of chemical pesticides have greatly promoted the development of agriculture. However, the harm of a large amount of chemical pesticides to the environment and higher animals cannot be ignored. On the one hand, it brings serious environmental pollution and becomes an important disease inducer, leading to irreversible and harmful genetic modification; These organisms have developed strong drug resistance. Plant secondary substances have good control effects on diseases, insect pests and weeds, are safe for humans and animals, do not pollute the environment, have no residues, are highly specific to diseases, insect pests and weeds, come from nature, are easily degraded, and are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12A01N47/44A01N35/06A01N43/90A01P7/04
CPCA01N35/06A01N43/90A01N47/44C07K14/43563A01N2300/00
Inventor 高希武杨丽文向敏刘晓岚张雷路瑶
Owner CHINA AGRI UNIV
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