NPF neuropeptide of cotton bollworm and helicoverpa assulta as well as encoding gene and application thereof

A gene and encoding technology, applied to the NPF neuropeptide and its encoding gene and application field of cotton bollworm and tobacco spodoptera, to achieve significant beneficial value, inhibit harm, and good compatibility

Inactive Publication Date: 2014-03-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows creators to grow crop varieties resistant against certain types of mothers or other predators without causing damage. It works through blocking specific genetic material called PNP proteins, preventing them from being released into their surroundings during reproduction. By doing this it prevents these species from infecting humans while also promoting plant growth.

Problems solved by technology

The technical problem addressed in this patents relating specifically to controlling mothers' infested crop plants during spring through developing novel pesticidal technology based upon specific molecules from certain proteins involved in neuronal signal transduction pathways.

Method used

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  • NPF neuropeptide of cotton bollworm and helicoverpa assulta as well as encoding gene and application thereof
  • NPF neuropeptide of cotton bollworm and helicoverpa assulta as well as encoding gene and application thereof
  • NPF neuropeptide of cotton bollworm and helicoverpa assulta as well as encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1, the acquisition of cotton bollworm NPF protein and its coding gene

[0056] 1. cDNA library construction

[0057] The library was constructed using the SMART cDNA Library Construction Kit. The construction process is as follows: Take 2 μL of total RNA (about 2 μg) from the brain of cotton bollworm larvae, add 1 μL of SMART IV Oligoncleotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGG-3) and 1 μL of CDS / 3'PCR primer (ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N) in sequence , and then add 2 μL of RNase-free deionized water. Gently mix and centrifuge, incubate at 72°C for 2min, cool on ice for 2min and centrifuge, add 2μL 5x first-strand synthesis buffer, 1μL DTT (20mM), 1μL dNTP mixture (10mM), 1μL MMLV reverse transcriptase to each tube , to a total volume of 10 μL. At the same time, make a positive control. Gently mix, centrifuge, and incubate in a PCR machine at 42°C for 1 h, and stop the synthesis of the first strand on ice, and the synthesis of the first ...

Embodiment 2

[0090] Embodiment 2, the acquisition of NPF protein of Spodoptera taba and its coding gene

[0091] Carry out embodiment 1 by replacing the cotton bollworm with tobacco armigera, and find a new gene derived from tobacco armigera, a spliced ​​form of the new gene can also encode the protein shown in sequence 1 of the sequence table (name it tobacco Spodoptera NPF1 protein, the sequence is completely consistent with the NPF1 protein of the cotton bollworm), another spliced ​​form of the new gene can encode the protein shown in sequence 6 of the sequence listing (named as the NPF2 protein of Spodoptera taba, as shown in the sequence listing 6). Correspondingly, the gene encoding NPF1 protein derived from Spodoptera taba was found, named as Spodoptera taba NPF1 gene, as shown in sequence 5 of the sequence listing (its open reading frame is sequence 5 from the 5' end of the sequence listing 70- 315 nucleotides). Correspondingly, the gene encoding NPF2 protein derived from Spodopt...

Embodiment 3

[0094] Embodiment 3, the construction of RNAi interference carrier

[0095] 1. Construction of cotton bollworm RNAi interference vector

[0096] 1. Extract the total RNA of the 5th instar cotton bollworm larvae and reverse transcribe it into cDNA. Using cDNA as a template, using NPFFor and NPFRev as templates, using LA taq as DNA polymerase, carry out PCR amplification, and recover the PCR amplification product.

[0097] NPFFor:5'-G CCCGG ATGCTGAACAAGAACATCG-3';

[0098] NPFRev:5'-C GGATCC TCATCTCCTTCTGGCGTAC-3'.

[0099] In NPFFor, the restriction endonuclease SmaI restriction endonuclease recognition sequence is underlined, and the restriction endonuclease EcoRI restriction endonuclease recognition sequence is marked in a box. NPFRev underlines the recognition sequence for restriction endonuclease BamHI, and the box marks the recognition sequence for restriction endonuclease HindIII.

[0100] PCR amplification system: LA PCR Buffer (Mg 2+ ) 2.5 μL, dNTP mix (each 1...

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Abstract

The invention discloses NPF neuropeptide of cotton bollworm and helicoverpa assulta as well as an encoding gene and application thereof. The protein is as follows (a), (b), (c) or (d), wherein (a) the protein is the protein consisting of amino acid indicated by sequence 1; (b) the protein consisting of amino acid indicated by sequence 3; (c) the protein consisting of amino acid indicated by sequence 6; (d) the protein with the amino acid being substituted, omitted and/or derived. The invention also relates to application of the NPF neuropeptide in protecting a substance for inhibiting the gene expression as follows (e) or (f): (e) cultivating an insect-resisting transgenosis plant; (f) preparing insect resistant agent. The substance inhibiting the gene expression can be used for cultivating the transgenosis insect-resisting crops, namely inhibiting the harm of the insect on the plant by interfering the feeding of the insect, and is pollution-free, free from transferring harmful substances into the natural enemy body of the insect, good in environmental compatibility and significant to the agricultural production.

Description

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Claims

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Application Information

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Owner CHINA AGRI UNIV
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