Apolygus lucorum vitellogenin and specific peptide chain, vector, strain and application thereof

Abstract

The invention discloses a DNA sequence used for encoding apolygus lucorum vitellogenin. The invention further discloses a specific peptide sequence capable of detecting an AlVg protein expression level by using Western-blot, and the expression difference of AlVg in apolygus lucorum on the protein level is analyzed by utilizing the specificity of the polypeptide. The invention further constructs an atlas of age in days after female adult eclosion and AlVg expression trend. The correlation of AlVg genes and protein expression levels in female adults with single female fecundity is investigated after the female adults are continuously bred on a variety of host plants, in order to construct a related prediction model, which has significant importance on prediction of the spawning potential of the apolygus lucorum. a positive correlation relation between the AlVg expression levels of female apolygus lucorum adults and the single female fecundity after eclosion for 7 days is defined. The specific peptide sequence disclosed by the invention can be used for observing and predicting the population dynamics of the apolygus lucorum in fields, and providing a foundation for research and development of new technology for monitoring field populations of the apolygus lucorum.

Description

technical field

[0001] The invention relates to the field of agricultural science and technology, in particular to vitellogenin of Lygus viridans, its specific peptide chain, carrier, bacterial strain and application. Background technique

[0002] Insect vitellogenin (Vg protein) is an important protein present in the body fluid of female adult insects. It is the precursor of insect vitellin and plays an important role in the development of insect eggs. The level of its content in female insects determines the Spawning potential size. Apolygus lucorum (Hemiptera: Miridae) belongs to the Miridae family of Hemiptera, and is the main pest on various economic crops including Bt cotton in my country. Due to the characteristics of polyphagia, fast movement, and concealed feeding, the development of accurate measurement and forecasting technology for the population of Lygus is facing severe challenges in agricultural production. Accurate forecasting of the laying capacity of Lygu...

Examples

Embodiment 1

[0043] Embodiment 1: the acquisition of AlVg full-length gene

[0044]The TRIzol method was used to extract the total RNA of Lygus japonica, and the total RNA samples with good electrophoretic patterns and OD260 / OD280 values ​​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, 0.5μl Oligo-dT18, 2μl 5× reaction buffer, 2μl 10mM dNTP, 40U RNasin, 200U SuperscriptⅡ, add ddH 2 0 to 10 μl; PCR reaction parameters: 42°C for 30 min, 75°C for 30 min, 4°C for 5 min. According to the known conserved sequence of insect Vg gene, primers AlVg-1F (5'-CGTTGGTAGCCGCATGAAC-3') and AlVg-1R (5'-GAACTGCTGCTGGAACTGCTG-3') suitable for PCR amplification were designed to obtain the conserved sequence of AlVg gene. For the sequence, refer to SEQ ID NO: 19 in the sequence listing. The PCR reaction system is: 2.5 μl 10× reaction buffer, 2 μl 25 mM Mg 2+ , 2 μl 10 mM dN...

Embodiment 2

[0046] Embodiment 2: AlVg protein-specific peptide chain cDNA clone

[0047] According to the full-length sequence of the AlVg gene, the cDNA sequence of the specific peptide chain of the AlVg protein is predicted in the national authoritative gene database National Center for Biotechnology Information (NCBI). The base is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, and Design PCR amplification primers AlVg-3F (5′-CATATGCAAATCCAAATCTCAACGT-3′) and AlVg-3R (5′-TCTAGATCAAGCTTTGCAGCGAG-3′) to obtain AlVg protein specific peptide chain gene sequence, PCR reaction system: 2.5μl 10× Reaction buffer, 2 μl 25mM Mg 2+ , 2 μl 10 mM dNTP, 0.5 μl Taq plus DNA polymerase, 1 μl 10 μM upstream primer AlVg-1-F, 1 μl 10 μM downstream primer AlVg-1-R, 1 μl cDNA template, add ddH 2 O to 25 μl; PCR reaction parameters: 94°C for 5 minutes; 94°C for 30s, 60.5°C for 30s, 72°C for 60s, 40 cycles; 72°C for 10 minutes. The results of electrophoresis showed that after PCR a...

Embodiment 3

[0049] Embodiment 3: Construction of AlVg protein-specific peptide chain DNA prokaryotic expression vector

[0050] The method for constructing the prokaryotic expression vector containing the AlVg protein-specific peptide chain gene T vector is as follows: the above-mentioned sequencing is correctly connected into the pGEM-TEasy vector and pCzn1 are both digested with restriction endonucleases NdeI and XbaI, and connected. The enzyme digestion system is: 1.0 μl of each of the two restriction enzymes, 2.0 μl of 10×Buffer buffer, 10.0 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 20 μl, and bathe in water at 37°C for 3h. The ligation system is: 5.0 μl of the recovered vector after enzyme digestion, 10.0 μl of the gene fragment, 1.0 μl of T4 DNA ligase, 2.0 μl of 10×Buffer, and ddH 2 Make up O to 20 μl, react at 16°C for 12-16 hours, and see the enzyme digestion map after successful ligation Figure 4 . After the ligation product was transfor...

technical field

[0001] The invention relates to the field of agricultural science and technology, in particular to vitellogenin of Lygus viridans, its specific peptide chain, carrier, bacterial strain and application. Background technique

[0002] Insect vitellogenin (Vg protein) is an important protein present in the body fluid of female adult insects. It is the precursor of insect vitellin and plays an important role in the development of insect eggs. The level of its content in female insects determines the Spawning potential size. Apolygus lucorum (Hemiptera: Miridae) belongs to the Miridae family of Hemiptera, and is the main pest on various economic crops including Bt cotton in my country. Due to the characteristics of polyphagia, fast movement, and concealed feeding, the development of accurate measurement and forecasting technology for the population of Lygus is facing severe challenges in agricultural production. Accurate forecasting of the laying capacity of Lygu...

Examples

Embodiment 1

[0043] Embodiment 1: the acquisition of AlVg full-length gene

[0044]The TRIzol method was used to extract the total RNA of Lygus japonica, and the total RNA samples with good electrophoretic patterns and OD260 / OD280 values ​​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, 0.5μl Oligo-dT18, 2μl 5× reaction buffer, 2μl 10mM dNTP, 40U RNasin, 200U SuperscriptⅡ, add ddH 2 0 to 10 μl; PCR reaction parameters: 42°C for 30 min, 75°C for 30 min, 4°C for 5 min. According to the known conserved sequence of insect Vg gene, primers AlVg-1F (5'-CGTTGGTAGCCGCATGAAC-3') and AlVg-1R (5'-GAACTGCTGCTGGAACTGCTG-3') suitable for PCR amplification were designed to obtain the conserved sequence of AlVg gene. For the sequence, refer to SEQ ID NO: 19 in the sequence listing. The PCR reaction system is: 2.5 μl 10× reaction buffer, 2 μl 25 mM Mg 2+ , 2 μl 10 mM dN...

Embodiment 2

[0046] Embodiment 2: AlVg protein-specific peptide chain cDNA clone

[0047] According to the full-length sequence of the AlVg gene, the cDNA sequence of the specific peptide chain of the AlVg protein is predicted in the national authoritative gene database National Center for Biotechnology Information (NCBI). The base is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, and Design PCR amplification primers AlVg-3F (5′-CATATGCAAATCCAAATCTCAACGT-3′) and AlVg-3R (5′-TCTAGATCAAGCTTTGCAGCGAG-3′) to obtain AlVg protein specific peptide chain gene sequence, PCR reaction system: 2.5μl 10× Reaction buffer, 2 μl 25mM Mg 2+ , 2 μl 10 mM dNTP, 0.5 μl Taq plus DNA polymerase, 1 μl 10 μM upstream primer AlVg-1-F, 1 μl 10 μM downstream primer AlVg-1-R, 1 μl cDNA template, add ddH 2 O to 25 μl; PCR reaction parameters: 94°C for 5 minutes; 94°C for 30s, 60.5°C for 30s, 72°C for 60s, 40 cycles; 72°C for 10 minutes. The results of electrophoresis showed that after PCR a...

Embodiment 3

[0049] Embodiment 3: Construction of AlVg protein-specific peptide chain DNA prokaryotic expression vector

[0050] The method for constructing the prokaryotic expression vector containing the AlVg protein-specific peptide chain gene T vector is as follows: the above-mentioned sequencing is correctly connected into the pGEM-TEasy vector and pCzn1 are both digested with restriction endonucleases NdeI and XbaI, and connected. The enzyme digestion system is: 1.0 μl of each of the two restriction enzymes, 2.0 μl of 10×Buffer buffer, 10.0 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 20 μl, and bathe in water at 37°C for 3h. The ligation system is: 5.0 μl of the recovered vector after enzyme digestion, 10.0 μl of the gene fragment, 1.0 μl of T4 DNA ligase, 2.0 μl of 10×Buffer, and ddH 2 Make up O to 20 μl, react at 16°C for 12-16 hours, and see the enzyme digestion map after successful ligation Figure 4 . After the ligation product was transfor...

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