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Acyrthosiphon pisum CCAP gene and ligand and receptor thereof, and application of acyrthosiphon pisum CCAP gene and ligand and receptor in aphid insect specific control agent

A CCAP-2-R, pea aphid technology, applied in the field of genetic engineering, can solve the problems of aggravating the outbreak of agricultural pests, low specificity of pesticides, hidden dangers of food safety, etc., and achieves the effect of good application prospects.

Active Publication Date: 2020-04-21
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, pea aphids have developed serious resistance to pesticides such as imidacloprid and acetamiprid on the market; pesticide residues seriously pollute the environment and pose hidden dangers to food safety; pesticides have low specificity and threaten natural enemies of harmful organisms. Increased risk of agricultural pest outbreaks

Method used

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  • Acyrthosiphon pisum CCAP gene and ligand and receptor thereof, and application of acyrthosiphon pisum CCAP gene and ligand and receptor in aphid insect specific control agent
  • Acyrthosiphon pisum CCAP gene and ligand and receptor thereof, and application of acyrthosiphon pisum CCAP gene and ligand and receptor in aphid insect specific control agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Obtaining the Open Reading Frame Sequence of Pea Aphid CCAP and Its Receptor

[0037] 1. Primer design and amplification of the open reading frame sequence of the pea aphid CCAP and its receptor:

[0038] Based on the genome and transcriptome database of the pea aphid, the CCAP and CCAP-R of Tribolium castaneum were used for TBLASTN search, the nucleotide sequences of CCAP and its receptor CCAP-R were predicted, and CCAP was designed and amplified The nested PCR specific primers are CCAP-1-F / CCAP-1-R (the first round of amplification primers) and CCAP-2-F / CCAP-2-R (the second round of amplification primers), the amplification The primers of CCAP-R are CCAP-R-1-F / CCAP-R-1-R (first round of amplification primers) and CCAP-R-2-F / CCAP-R-2-R (second round of amplification primers) primers), the primer sequences are as follows:

[0039]

[0040] The total RNA of the pea aphid was extracted with an RNA extraction kit, and reverse-transcribed into cDNA with a rev...

Embodiment 2

[0053] Example 2: Determination of the Binding Ability of the Ligand Polypeptide to Its Receptor Based on the Method of Intracellular Calcium Ion Flux Detection 1. Construction of the CHO (Chinese Hamster Ovary Cell) Cell Expression Plasmid of Pea Aphid CCAP-R

[0054] Construction of pcDNA3.1-CCAP-R expression vector: add Kpn I and Xba I restriction sites to the ORF of CCAP-R by PCR, and use Kpn I and Xba I to perform restriction digestion, pcDNA3.1(+) Plasmids were also digested with Kpn I and Xba I and then ligated. After ligation, transform according to the above-mentioned transformation method. After the positive identification of the bacterial liquid was correct, the plasmid was extracted with a plasmid extraction kit, and the CCAP-R fragment was constructed. pcDNA3.1(+) plasmid.

[0055] Two, the above-mentioned pcDNA3.1 (+) plasmids obtained by constructing the CCAP-R fragments are co-transfected respectively, and are operated according to the following steps:

[0056...

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Abstract

The invention discloses an acyrthosiphon pisum CCAP gene of which the sequence is shown as SEQ ID NO: 9. The invention further discloses an amplification method of the gene, nested PCR is adopted, andprimers are CCAP-1-F / CCAP-1-R and CCAP-2-F / CCAP-2-R. The invention further discloses an acyrthosiphon pisum CCAP ligand, the amino acid sequence of the acyrthosiphon pisum CCAP ligand is shown as SEQID NO: 11, and nucleotide sequences for encoding the amino acid sequence are shown as SEQ ID NO: 10. The invention further discloses an open reading frame sequence of an acyrthosiphon pisum CCAP receptor, and the open reading frame sequence is shown as SEQ ID NO: 12. An amplification method of the open reading frame sequence of the acyrthosiphon pisum CCAP receptor is further disclosed, and a primer pair CCAP-R-1-F / CCAP-R-1-R and CCAP-R-2-F / CCAP-R-2-R is adopted. The invention further discloses an application of the acyrthosiphon pisum CCAP gene and the ligand and receptor thereof in preparation of aphid insecticides and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a pea aphid CCAP gene, its ligand, receptor and its application in a specific control agent for aphids. Background technique [0002] Pea aphid (Acyrthosiphonpisum) is an insect belonging to the family Aphidaceae and the genus Aphid. It is an important pest of many leguminous crops and forages. It not only directly feeds on host plants, causing serious economic losses, but also transmits a variety of plant viruses. As a model insect for ecological research, the pea aphid has many characteristics such as complex life cycle, diverse reproductive modes, phenotypic plasticity, and complex symbiotic relationship with bacteria, and has become a research object of interest to many scientists. At present, pea aphids have developed serious resistance to pesticides such as imidacloprid and acetamiprid on the market; pesticide residues seriously pollute the environment and pos...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K7/06C07K14/435A01N63/14A01N37/46A01P7/04
CPCA01N37/46C07K7/06C07K14/43563C12N15/10
Inventor 石岩杨春红刘同先
Owner QINGDAO AGRI UNIV
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