Spodoptera exigua vitellogenin receptor, specific peptide chain, vector, strain and application thereof

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...

Examples

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] Embodiment 2: SEVgR specific peptide chain cDNA clone

[0040] According to the measured full-length sequence of the SEVgR gene, DNAMAN software was used to predict the cDNA sequence of the SEVgR specific peptide chain, 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: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1 min; 72°C for 10 minutes. The results of electrophoresis showed that after PCR amplification and splicing, a band consistent with the expected size of 786bp was obtained ( image 3 ). The PCR product was separated by 1.2% aga...

Embodiment 3

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

[0043] The method for constructing the prokaryotic expression vector containing the SEVgR-specific peptide chain gene T vector is as follows: link the correctly sequenced PCR product of Example 2 into the expression vector pET15B through the cloning sites Nde I and Xho I, and verify whether a recombinant plasmid is formed by enzyme digestion. The digestion system is: 0.25 μl each of the two restriction enzymes (NdeI and XbaI), 1.0 μl of 10×Buffer buffer, 3 μl of plasmid, and ddH 2 Make up O to 10 μl, and bathe in water at 37°C for 3h. For the enzyme digestion map after successful connection, see Figure 4 . Transform the correctly identified recombinant plasmid named pET15B-SEVgR into 100 μl Escherichia coli Arctic Express (Zhongding Biological Company) competent cells, put it on ice for 20 minutes, heat shock at 42°C for 90 seconds, quickly put it in ice for 5 minutes, and add...

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...

Examples

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] Embodiment 2: SEVgR specific peptide chain cDNA clone

[0040] According to the measured full-length sequence of the SEVgR gene, DNAMAN software was used to predict the cDNA sequence of the SEVgR specific peptide chain, 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: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1 min; 72°C for 10 minutes. The results of electrophoresis showed that after PCR amplification and splicing, a band consistent with the expected size of 786bp was obtained ( image 3 ). The PCR product was separated by 1.2% aga...

Embodiment 3

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

[0043] The method for constructing the prokaryotic expression vector containing the SEVgR-specific peptide chain gene T vector is as follows: link the correctly sequenced PCR product of Example 2 into the expression vector pET15B through the cloning sites Nde I and Xho I, and verify whether a recombinant plasmid is formed by enzyme digestion. The digestion system is: 0.25 μl each of the two restriction enzymes (NdeI and XbaI), 1.0 μl of 10×Buffer buffer, 3 μl of plasmid, and ddH 2 Make up O to 10 μl, and bathe in water at 37°C for 3h. For the enzyme digestion map after successful connection, see Figure 4 . Transform the correctly identified recombinant plasmid named pET15B-SEVgR into 100 μl Escherichia coli Arctic Express (Zhongding Biological Company) competent cells, put it on ice for 20 minutes, heat shock at 42°C for 90 seconds, quickly put it in ice for 5 minutes, and add...

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