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Chilo suppressalis ryanodine receptor gene CsRyR, encoded protein thereof and application of chilo suppressalis ryanodine receptor gene CsRyR

A kind of technology of nitin and nicotine receptor of Diplostis chinensis, which is applied in the fields of application, receptor/cell surface antigen/cell surface determinant, genetic engineering, etc.

Inactive Publication Date: 2012-08-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although it has long been discovered that there are a large number of RyR sites in insect muscle cells through physiological and biochemical methods (Vazquez-Martinez et al., 2003), compared with the more systematic and complete research on mammalian RyR, insect RyR molecules The level of research has been almost blank for a long time, and only the full-length cDNA of the RyR gene of Drosophila melanogaster has been cloned (Takeshima et al., 1994) and the study of channel dynamics has been carried out (Xu et al. , 2000)

Method used

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  • Chilo suppressalis ryanodine receptor gene CsRyR, encoded protein thereof and application of chilo suppressalis ryanodine receptor gene CsRyR
  • Chilo suppressalis ryanodine receptor gene CsRyR, encoded protein thereof and application of chilo suppressalis ryanodine receptor gene CsRyR
  • Chilo suppressalis ryanodine receptor gene CsRyR, encoded protein thereof and application of chilo suppressalis ryanodine receptor gene CsRyR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Cloning of the CsRyR Gene of Chipotle Borer Nitin Receptor

[0062] 1. Homogenize the 4th instar Chilo borer larvae with the Trizol method to extract total RNA and transcribe it into cDNA:

[0063] Use TransScript TM II Reverse Transcriptase for reverse transcription experiments.

[0064] reaction system:

[0065]

[0066] Reaction conditions: 65°C, 5 minutes, immediately place on ice for 2 minutes, then add the following components:

[0067]

[0068] Reaction conditions: 30°C, 10min 42°C, 30min 95°C, 5min

[0069] 2. PCR amplification of the full-length gene sequence

[0070] PCR amplification using Roche's Expand Long Range dNTPack

[0071] reaction system:

[0072]

[0073] Reaction conditions:

[0074] The final product is a full-length sequence (SEQ ID NO: 1, ie Sequence Listing 1) containing Not1 and Apa1 restriction endonucleases at both ends.

Embodiment 2

[0076] Expression of the receptor protein CsRyR of Chilo suppressalis in HEK-293 cell line

[0077] 1. Cloning the CsRyR gene sequence (SEQ ID NO: 1) obtained by cloning into the pcDNA3 expression vector and preparing the Not I digestion reaction of the pcDNA3 expression vector

[0078] reaction system:

[0079]

[0080] Reaction condition: 37℃ 4hours

[0081] Use TaKaRa DNA Fragment Purification Kit to purify the above digestion product; obtain pCDNA3-Not I.

[0082] Apa I digestion reaction

[0083] reaction system:

[0084] pCDNA3-Not I 42.5ul

[0085] 10×L Buffer 5ul

[0086] Apa I (10U / ul) 2.5ul

[0087] Reaction condition: 37℃ 4hours

[0088] Use the TaKaRa Agarose Gel DNA Purification Kit to cut the gel and recover the above digested products.

[0089] The sequence obtained in Example 1 was ligated into the above restriction vector with ligase to obtain the expression vector pcDNA3-CsRyR.

[0090] 2. Transfection of HEK-293 cell line and monoclonal cell cult...

Embodiment 3

[0093] Calcium ion responses of HEK-CsRyR cell line to insecticides

[0094] The expressed CsRyR cells (HEK-CsRyR cells) were transferred to 12mm coverslips 48h in advance, washed twice with DPBS, then loaded with a mixture of Fura-2AM (final concentration 5M) and DMEM for 30min, and then washed twice with Ringer buffer. After the second pass, the fluorescence intensity of the emission wavelength of 510nm under the excitation wavelength of 340nm and 380nm respectively was measured on the ERP calcium ion imaging system of PTI company, and the ratio between the two can be converted into the absolute concentration of intracellular calcium ion.

[0095] The cells to be tested (HEK-CsRyR cells) were added different concentrations of agonists and insecticides under buffer perfusion to record the changes in the ratio of fluorescence intensity. details as follows:

[0096] 1. At room temperature, use Ringer Buffer to perfuse the bath tank storing slides at a rate of 1 mL / min, then us...

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Abstract

The invention discloses a chilo suppressalis ryanodine receptor gene CsRyR, which is used for encoding a nucleotide sequence with a chilo suppressalis ryanodine receptor protein CsRyR, wherein the nucleotide sequence has at least 70 percent of homology with a nucleotide sequence shown as SEQ ID NO:1. The invention further discloses a protein encoded by using the chilo suppressalis ryanodine receptor gene CsRyR. The protein has an amino acid sequence shown as SEQ ID No:2. The chilo suppressalis ryanodine receptor gene CsRyR is used for screening and / or evaluating a substance for regulating and controlling the activity of a chilo suppressalis ryanodine receptor.

Description

technical field [0001] The invention relates to the fields of molecular biology, cell biology and pesticide science, and mainly relates to a ryanodine receptor gene (nucleic acid sequence) expressed in Chilo suppressalis and its encoded protein. The present invention also relates to the preparation method and application of the protein and receptor gene (nucleic acid sequence). Background technique [0002] The name of the ryanodine receptor (RyR) comes from the plant alkaloid insecticide ryanodine isolated from a leguminous plant of Rhyania speciosa in South America. Studies have shown that ryanodine produces insecticidal effects by acting on RyR (Fill and Copello, 2002). However, due to its complex structure, difficulty in transformation, high cost and high toxicity, its use as an insecticide is limited. After long-term research, Nihon Nohyaku finally discovered the insecticidal activity of phthalic diamides and commercialized flubendiamide (Tohnishi et al., 2005). Almo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/705C12N15/63C12N5/10C12N1/19C12Q1/02
Inventor 黄佳吴顺凡叶恭银
Owner ZHEJIANG UNIV
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