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Apoptosis gene for reducing egg laying amount of laodelphax striatellus and application thereof

A technology of SBPH and cells, applied in the field of apoptosis gene and its application

Active Publication Date: 2020-03-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that there are certain defects in the existing methods for preventing and treating SBPH

Method used

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  • Apoptosis gene for reducing egg laying amount of laodelphax striatellus and application thereof
  • Apoptosis gene for reducing egg laying amount of laodelphax striatellus and application thereof
  • Apoptosis gene for reducing egg laying amount of laodelphax striatellus and application thereof

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Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0035] The cloning method of the gene Lsdark which reduces the amount of Laodelphax striatellus includes the following steps:

[0036] S10. Extract the total RNA (ribonucleic acid) of the female adult Laodelphax striatellus, and synthesize the first strand of cDNA using the total RNA of Laodelphax striatellus as a template;

[0037] The specific process is to take 40-50 ovaries of female adult Laodelphax striatellus, extract total RNA using TRizoL method, and use total RNA as a template to synthesize the first strand of cDNA by reverse transcription.

[0038] S11. Using the synthesized first strand of Laodelphax striatellus cDNA as a template, PCR amplification is performed through the upstream primer P1 and the downstream primer P2;

[0039] The specific process is to obtain the gene fragment sequence from the Laodelphax striatellus genome, and after homology comparison in the NCBI database, it is predicted to be the Lsdark gene of Laodelphax striatellus, and the upstream primer P1 (S...

specific Embodiment 2

[0047] The method for synthesizing dsRNA of the gene Lsdark, which reduces the amount of Laodelphax striatellus, includes the following steps:

[0048] S20. Extract total RNA from the adult female Laodelphax striatellus, and synthesize the first strand of cDNA using the total RNA of Laodelphax striatellus as a template;

[0049] S21. Using the first strand of Laodelphax striatellus cDNA synthesized in step S1 as a template, PCR amplification is carried out with the upstream primer P3 and the downstream primer P4, and the PCR amplified products are separated by agarose gel electrophoresis at a concentration of 1.2%. The gel was cut under UV light and recovered using AxyPrep DNA Gel Recovery Kit:

[0050] The specific process is to design the upstream primer P3 (SEQ ID NO. 6) and downstream primer P4 (SEQ ID NO. 7) based on the verified Lsdark gene sequence using Primer-Blast in the NCBI database.

[0051] Upstream primer P3 (SEQ ID NO.6):

[0052] 5′-taatacgactcactatagggGTGAGCGATTTGCCTT...

Embodiment 3

[0078] The method of feeding Laodelphax striatellus using the gene fragment dsRNA mixed with feed is as follows:

[0079] S30. Evenly mix the Laodelphax striatellus feed with the gene fragment dsRNA (concentration of 4000ng / μL);

[0080] S31. Prepare multiple double-pass glass tubes, seal one end of the double-pass glass tube with gauze and rubber bands, and use an electric trap to suck 2-3 instar Laodelphax striatellus nymphs into the double-pass glass tube;

[0081] S32. Seal the other end of the two-way glass tube with Parafilm sealing film, take another sealing film, and seal the mixture of Laodelphax striatellus feed and apoptotic dsRNA between the two sealing films.

[0082] Use a pipette to suck 100 μL of the dsRNA mixture of Laodelphax striatellus feed and Lsdark gene fragment (treatment group) or 100 μL of the dsGFP mixture of Laodelphax striatellus feed and control GFP gene (control group) and drop them in the center of the two parafilms.

[0083] S33. Place the double-pass gl...

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Abstract

The invention discloses an apoptosis gene for reducing the egg laying amount of laodelphax striatellus and application of the apoptosis gene. The gene segment sequence for reducing the egg laying amount of laodelphax striatellus is shown as SEQ ID NO.1; and an amino acid sequence is shown as SEQ ID NO.2. According to the invention, the apoptosis genes capable of causing significant reduction of the egg laying amount of laodelphax striatellus is screened out; the dsRNA sequence of the laodelphax striatellus apoptosis gene segment, which is synthesized by a molecular biological technique, is shown as SEQ ID NO.5; the dsRNA of the apoptosis gene segment is fed with the laodelphax striatellus after being mixed with feed; and the synthesized dsRNA of the laodelphax striatellus apoptosis gene segment can effectively silence genes, well resist the degradation of RNA enzymes, reduce the reproductive capacity of the laodelphax striatellus, and reduce population of the laodelphax striatellus, and therefore, with the apoptosis gene adopted, the laodelphax striatellus can be controlled.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to an apoptosis gene for reducing the amount of eggs laid by Laodelphax striatellus and its application. Background technique [0002] Laodelphax striatellus is the main pest in my country's grain areas. In addition to directly feeding on food crops, it can also spread a variety of crop viruses and indirectly harm crops, causing a 30-50% reduction in food crop production or even no harvest every year. [0003] At present, the control of Laodelphax striatellus is mainly based on chemical pesticides, but Laodelphax striatellus has developed different levels of resistance to various commonly used insecticides, resulting in frequent outbreaks. In the 1970s, organochlorine pesticides were banned from the control of Laodelphax striatellus due to environmental pollution, high toxicity and high resistance. From the 1980s to the 1990s, some organophosphorus pesticides and carbamate pesticides were use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/113A01N63/60A01P7/04
CPCC07K14/43563C12N15/113A01N57/16C12N2310/14
Inventor 郭燕
Owner SUN YAT SEN UNIV