Neuropeptide natalisin and its receptor gene and its application in specific control agent of Bactrocera dorsalis
A technology of natalisin-2-r and natalisin-2-f, which is applied in the field of genetic engineering and achieves good application prospects
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Embodiment 1
[0040] Example 1, Obtaining the Open Reading Frame Sequence of Bacteralis dorsalis Neuropeptide Natalisin and Its Receptor
[0041] 1. Primer design and amplification of the open reading frame sequence of Bacteralis dorsalis neuropeptide Natalisin and its receptor
[0042] Based on genome and transcriptome data, using bioinformatics methods, after repeated analysis and comparison of Drosophila Natalisin (Genbank No. NM001170137) and its Natalisin receptor (Genbank No. CG6515), the neuropeptide Natalisin and its The nested PCR specific primers of the acceptor, the sequence is as follows:
[0043]
[0044] The total RNA of B. dorsalis was extracted with an RNA extraction kit, and reverse transcribed into cDNA with a reverse transcription kit.
[0045]Amplification of Bacteralis dorsalis neuropeptide Natalisin sequence: 50ul reaction system containing 24μL deionized water, 20μL 2×PrimeSTAR Max Premix, primers Natalisin-1-F (as shown in SEQ ID NO: 1 in the sequence listing), N...
Embodiment 2
[0060] Example 2. Determination of the Binding Ability of Bacteralis dorsalis Neuropeptide Natalisin and Its Receptor Based on the Intracellular Calcium Ion Flow Detection Method
[0061] 1. Construction of Bacteralis dorsalis Natalisin receptor CHO (Chinese hamster ovary cell) cell expression plasmid
[0062] The Bacteralis dorsalis neuropeptide Natalisin acceptor plasmid and pcDNA3.1 (+) plasmid connected on the T carrier recovered in Example 1 were connected (T4 DNA ligase) after NotI single enzyme digestion respectively, and connected Then refer to the transformation method in Example 1 for transformation, and after the positive identification of the bacterial liquid is correct (PCR detection with the universal primer T7 / BGH, T7: TAATACGACTCACTATAGGG, BGH: TAGAAGGCACAGTCGAGG), the plasmid is extracted with a plasmid extraction kit, and the construction is obtained pcDNA3.1(+) plasmid with NTLR fragment.
[0063] Co-transfection:
[0064] 1) Prepare 1.5mL serum-free mediu...
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