Neuropeptide cv-snpf and its receptor in Plutella xylostella xylostella and its application in increasing trehalose content in Plutella xylostella

A technology of Plutella xylostella and neuropeptide, which is applied in the fields of molecular biology and genetic engineering, can solve problems that do not involve regulation mechanisms and the like, and achieve the effect of increasing the content of trehalose

Active Publication Date: 2020-09-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, research on physiological regulation in parasitic systems mainly focuses on immunity, growth and development, and nutrient metabolism. There are only sporadic reports on other neuroendocrine studies, such as some neuropeptide genes of the host, allatostatin, The expression of allatotropin and prothoraciotropichormone (PTTH) changes significantly before and after the parasitoid parasitoid, but none of them involve the regulation mechanism

Method used

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  • Neuropeptide cv-snpf and its receptor in Plutella xylostella xylostella and its application in increasing trehalose content in Plutella xylostella
  • Neuropeptide cv-snpf and its receptor in Plutella xylostella xylostella and its application in increasing trehalose content in Plutella xylostella
  • Neuropeptide cv-snpf and its receptor in Plutella xylostella xylostella and its application in increasing trehalose content in Plutella xylostella

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Experimental program
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Effect test

Embodiment 1

[0033] 1. Acquisition of the ORF sequences of the Cv-sNPF gene of Plutella xylostella xylostella and the Px-sNPFR gene of Plutella xylostella

[0034] (1) Collect Plutella xylostella larvae and Plutella xylostella larvae: take the Plutella xylostella about 5-6 days after parasitization, and dissect it in insect culture medium under a microscope. Tear a hole first to facilitate the larvae to slide out, add new medium, wash several times, discard the washing liquid, add pre-cooled 1ml TRIzol, grind and store at -80°C for later use; take the diamondback moth that has grown to the 4th instar Three larvae were washed with alcohol and placed in 1ml TRIzol, ground and stored at -80°C for later use.

[0035] (2) Using the TRIzol method to extract the total RNA of Plutella xylostella and Plutella xylostella, and then use the kit SuperScript TM II Reverse Transcritase (Invitrogen) was used to synthesize cDNA first strand.

[0036] (3) Based on the transcriptome data of Plutella xylos...

Embodiment 2

[0055] 1. Synthesis of Cv-sNPF mature peptide and preparation of specific antibody

[0056] Protean software was used to analyze the hydrophilicity and secondary structure of the peptide precursor protein sequence, and a specific 10-20aa fragment was selected for synthesis (as shown in SEQ ID NO.5). The synthetic sequence is shown in Table 2. The synthesized short peptide was sent to Shanghai Sangon Biological Co., Ltd. for synthesis, and a total of 5 mg was synthesized with a purity of >90%. Antibodies are prepared using a synthetic polypeptide (such as SEQ ID NO.5) as an antigen.

[0057] Table 2 Design and synthesis of short peptide sequences

[0058]

[0059] 2. The localization of Cv-sNPF in the nervous system of the larvae of Plutella xylostella

[0060] 2.1 Dissection of different tissues

[0061] Take 10-20 larvae of the same age and the same growth and development of Plutella xylostella, dissect the brain tissue under a stereo microscope, and put it in cold PBS ...

Embodiment 3

[0080] 1. Screening of stably transfected cell lines

[0081] 1.1 Construction of eukaryotic expression vector

[0082] Using the primers designed as follows (Table 3), PCR amplifies the Px-sNPFR gene fragment with restriction sites, and through conventional methods of constructing vectors such as double digestion, ligation, and transformation, the Px-sNPFR is connected to the eukaryotic expression Vector pcDNA3.1.

[0083] Table 3 Construction of primers for eukaryotic expression vectors with restriction sites

[0084]

[0085] 1.2 Cell culture, transfection and screening

[0086] HEK293 cells were cultured using DMEM high-glucose medium produced by Invitrogen, supplemented with inactivated 10% fetal bovine serum (Fetal Bovine Serum, FBS) medium. Cells were cultured at 37°C, 5% CO2 incubator (ThermoScientific). The cell subculture was carried out in a sterilized bench, and it was sterilized by ultraviolet light for more than 20 minutes before starting. During operatio...

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Abstract

The invention discloses a cotesia-vestalis neuropeptide Cv-sNPF, a receptor thereof and application to the improvement of trehalose content in the body of plutella xylostella. According to the cotesia-vestalis neuropeptide Cv-sNPF and the receptor thereof, the cotesia-vestalis neuropeptide Cv-sNPF and the receptor Px-sNPFR thereof hosting in the body of the plutella xylostella are discovered in acotesia vestalis-plutella xylostella parasitic system for the first time; the cotesia-vestalis neuropeptide Cv-sNPF not only can be bound with the receptor Px-sNPFR in the body of the plutella xylostella, but also can be used for improving the trehalose content in the body of the plutella xylostella.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, and in particular to the neuropeptide Cv-sNPF of Braconis xylostella xylostella and its receptor and its application in increasing the trehalose content in the body of diamondback moth. Background technique [0002] Neuropeptides are an important class of small molecular substances that transmit information, affecting almost all physiological processes in organisms, existing as neuromodulators in the central nervous system, and acting as neurohormones in the blood. [0003] short neuropeptide F (sNPF) is an important peptide substance in invertebrates, which is involved in the regulation of feeding, growth and development, movement, clock rhythm, learning and memory and other physiological processes. It is an orthologous protein of vertebrate neuropeptide Y (NPY), and its sequence is similar, and it plays a regulatory role by binding to G protein-coupled recepto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K14/705
CPCC07K14/43563C07K14/705
Inventor 陈学新邹佳妮王泽华时敏黄健华
Owner ZHEJIANG UNIV
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