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A kind of beet armyworm vitellogenin receptor, its specific peptide chain, carrier, bacterial strain and application

A technology of vitellogenin, beet armyworm, applied in the field of agricultural science

Active Publication Date: 2019-10-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on Spodoptera exigua Vitellogenin (SEVgR) has not been reported at home and abroad. Therefore, the full-length DNA sequence of SEVgR was cloned, the specific peptide chain of SEVgR gene was isolated and purified, and its polyclonal antibody was prepared. , providing a good method guarantee for revealing the occurrence regularity and regulation function of vitellin receptor in beet armyworm

Method used

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  • A kind of beet armyworm vitellogenin receptor, its specific peptide chain, carrier, bacterial strain and application
  • A kind of beet armyworm vitellogenin receptor, its specific peptide chain, carrier, bacterial strain and application
  • A kind of beet armyworm vitellogenin receptor, its specific peptide chain, carrier, bacterial strain and application

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Experimental program
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Embodiment 1

[0036] Embodiment 1: Obtaining of SEVgR full-length gene

[0037] The total RNA of beet armyworm was extracted by TRIzol method, and the total RNA samples with good electrophoretic pattern and OD260 / OD280 value between 1.8 and 2.0 were selected and combined for mRNA purification, and the first-strand cDNA was synthesized by reverse transcription. The PCR reaction system was: 0.5 μg total RNA, 2.5μl Oligo-dT18, 5μl MMlv Buffer, 5μl 10mM dNTP, 40U RNasin, 1μl MMlv reverse transcriptase (reverse transcriptase), add ddH 2 O to 25 μl; PCR reaction parameters: 42°C for 1h, 95°C for 10min. According to the conserved sequence of the known insect VgR gene, primers SEVgR-3396F (5'-CGAAGAGGACTGTGATGAACCT-3') and SEVgR-4845R (5'-GACAAATGTGTTGGCGTCAA-3') suitable for PCR amplification were designed to obtain the conserved sequence of the SEVgR gene. For the sequence, refer to SEQ ID NO: 11 in the sequence listing, the PCR reaction system is: 5 μl 10× reaction buffer, 5 μl 25mM Mg 2+, 8μl...

Embodiment 2

[0039] Example 2: SEVgR-specific peptide chain cDNA cloning

[0040] According to the measured full-length sequence of SEVgR gene, the cDNA sequence of SEVgR-specific peptide chain was predicted by DNAMAN software, and PCR amplification primers SEVgR-3F (5′-TGTCATGAAACTGAGTTCATGTG-3′) and SEVgR-3R (5′-GCATGAGAACTGATCCTTGGACG- 3′) to obtain the SEVgR specific peptide chain gene sequence, the PCR reaction system is: 5 μl 10× reaction buffer, 5 μl 25mM Mg 2+ , 8 μl 10 mM dNTP, 0.5 μl Takara LA DNA polymerase, 1 μl 10 μM upstream primer SEVgR-3F, ​​1 μl 10 μM downstream primer SEVgR-3R, 1 μl cDNA template prepared in Example 1, add ddH 2 O to 50 μl; PCR reaction parameters are: 94°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 1 min, 35 cycles; 72°C for 10min extension. The electrophoresis results showed that after PCR amplification and splicing, a band consistent with the expected size of 786 bp was obtained ( image 3 ). The PCR product was separated by 1.2% agarose gel el...

Embodiment 3

[0042] Example 3: Construction of SEVgR-specific peptide chain DNA prokaryotic expression vector

[0043] The construction method of the prokaryotic expression vector containing the SEVgR-specific peptide chain gene T vector is as follows: the PCR product sequenced correctly in Example 2 is connected to the expression vector pET15B through the cloning sites Nde I and Xho I, and it is verified by enzyme digestion whether a recombinant plasmid is formed. The digestion system was as follows: 0.25 μl of each of the two restriction enzymes (NdeI and XbaI), 1.0 μl of 10× Buffer, 3 μl of plasmid, and ddH 2 O supplemented to 10 μl, water bath at 37°C for 3h. See the enzyme cleavage map after successful ligation Figure 4 . The correctly identified recombinant plasmid named pET15B-SEVgR was transformed into 100 μl E. coli Arctic Express (Zhongding Biotechnology) competent cells, placed on ice for 20 min, heat-shocked at 42°C for 90 s, quickly placed in ice for 5 min, and added 600 μl...

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Abstract

The invention discloses a DNA sequence for encoding a spodoptera exigua vitellogenin receptor. The invention discloses a spodoptera exigua vitellogenin receptor. The invention also discloses a specific peptide chain of the spodoptera exigua vitellogenin receptor, and also discloses a preparation method for the specific peptide chain of the spodoptera exigua vitellogenin receptor. The invention also discloses a polyclonal antibody of the vitellogenin receptor polypeptide and a preparation method thereof. The specific antibody of polypeptide can be utilized to analyze the tissue distribution of the vitellogenin receptor in spodoptera exigua and differential expression on the protein level. The invention can be used for rapid detection of the protein content of a spodoptera exigua vitellogenin receptor and can provide the material basis and technical support for expression regulation and other molecular biology study.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a vitellogenin receptor of beet armyworm, its specific peptide chain, carrier, bacterial strain and application. Background technique [0002] The beet armyworm Spodoptera exigua (Hübner) belongs to Lepidoptera Noctuidae. It is an important polyphagous pest with worldwide distribution, which can seriously damage many important economic crops. At present, the control of beet armyworm in agricultural production is facing severe challenges. Due to its strong reproductive ability and high pesticide resistance, it is urgent to develop new pollution-free prevention and control methods to reduce the use of chemical pesticides. The vitellogenin receptor VgR is the main receptor that mediates the endocytosis of insect vitellogenin, plays a crucial role in the maturation of insect ovaries, and is also a potential target for studying pest control. Carrying out research on...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/705C12N15/70C12N5/10C12N1/21C07K16/28G01N33/68C12R1/19
Inventor 赵静孙洋肖留斌谭永安柏立新
Owner JIANGSU ACAD OF AGRI SCI
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