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Vitellogenin, its specific peptide chain, carrier, bacterial strain and application

A vitellogenin and green Lygus technology, applied in the field of agricultural science, can solve problems such as lack of protein expression detection methods

Inactive Publication Date: 2017-11-14
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The use of Vg content to develop pest population forecasting is a new method for Alygus lucorum Vitellogenein (AlVg) protein expression, which is relatively scarce. The relationship between the relative expression level and its egg production has not been reported at home and abroad

Method used

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  • Vitellogenin, its specific peptide chain, carrier, bacterial strain and application
  • Vitellogenin, its specific peptide chain, carrier, bacterial strain and application
  • Vitellogenin, its specific peptide chain, carrier, bacterial strain and application

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Experimental program
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Effect test

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, 200USuperscriptⅡ, 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 dNT...

Embodiment 2

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

[0047] According to the full-length sequence of the AlVg gene in the National Center for Biotechnology Information (NCBI), an authoritative international gene database, the cDNA sequence of the specific peptide chain of the AlVg protein is predicted, the base is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, and PCR is designed Amplify primers AlVg-3F (5′-CATATGCAAATCCAAATCTCAACGT-3′) and AlVg-3R (5′-TCTAGATCAAGCTTTGCAGCGAG-3′) to obtain AlVg protein specific peptide chain gene sequence, PCR reaction system: 2.5 μl 10× reaction buffer Solution, 2μl 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...

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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Abstract

The invention discloses a DNA sequence for encoding the vitellogenin of the green ligus bug. The invention also discloses a specific polypeptide sequence capable of detecting the expression of AlVg protein by Western-blot, and using the polypeptide to specifically analyze the differential expression of AlVg at the protein level in Lygus chlorosis. The present invention also constructs a map of the age of female adults after eclosion and the expression trend of AlVg. By investigating the relationship between the expression of AlVg gene and protein in female adults after continuous feeding of various host plants, and the number of eggs laid by a single female, the construction of a correlation prediction model is of great significance for predicting the egg-laying potential of Lygus aeruginosa. The present invention clarifies that there is a positive correlation between the expression level of AlVg of female adults of Lygus lyridus 7 days after eclosion and the amount of eggs laid by a single female. It provides a basis for the research and development of new technologies for field population monitoring of Lygus spp.

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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/70C12N1/21C12R1/19
Inventor 孙洋肖留斌柏立新谭永安赵静戴瀚洋
Owner JIANGSU ACAD OF AGRI SCI
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