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Apolygus lucorum cell nucleus hormone receptor E75, coding sequence thereof, carrier and bacterial strain

A technology of nuclear hormones and green lygus, applied in the field of agricultural science, can solve problems such as the rise of green lygus

Active Publication Date: 2017-08-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, due to the large-scale planting of transgenic Bt cotton and the adjustment of the agricultural industrial structure, the population of Lygus spp. has increased sharply, and the long-term dependence on chemical pesticide control has led to the increasing resistance of Lygus spp. The prevention and control of the green Lygus is facing severe challenges, and it is urgent to develop new pollution-free prevention and control methods to reduce the use of chemical pesticides

Method used

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  • Apolygus lucorum cell nucleus hormone receptor E75, coding sequence thereof, carrier and bacterial strain
  • Apolygus lucorum cell nucleus hormone receptor E75, coding sequence thereof, carrier and bacterial strain
  • Apolygus lucorum cell nucleus hormone receptor E75, coding sequence thereof, carrier and bacterial strain

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Experimental program
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Embodiment 1

[0029] Embodiment 1: Acquisition of the nuclear hormone receptor E75 gene (AlE75 gene) of Lygus green bug

[0030] The TRIzol method was used to extract the total RNA of Lygus japonica, 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: 10 μl Total RNA, 4.0 μl 5X reaction buffer (Sigma), 2 μl 10 mmol dNTP, 1 μl ribonucleotide inhibitor (Sigma), 2 μl reverse transcriptase (Sigma), add ddH 2 O to a reaction volume of 20 μl. The parameters of the PCR reaction were: warm bath at 42°C for 60 minutes, and warm bath at 72°C for 10 minutes. Primers AlE75-F (5'-CATTATGGWGTYTAYAGYTGTG-3') and AlE75-R (5' -AGHAGTTCATTCCAVCCTGCTC-3'), the above-mentioned primers are degenerate primers to obtain the conserved sequence of Lygus nuclei hormone receptor E75, the conserved sequence is shown in SEQ...

Embodiment 2

[0039] Embodiment 2: Construction of AlE75 gene prokaryotic expression vector

[0040] The method for constructing the prokaryotic expression vector containing the AlE75 gene T vector is as follows: the above sequenced correct fragment of Lygus lucidum nuclear hormone receptor E75 is connected to the target gene of the pMD18-T vector and the pCzn1 vector with the restriction endonuclease Ndel and Xbal double enzyme digestion, for large fragment ligation ( figure 2 ). The enzyme digestion system is: 0.25 μl of each of the two restriction endonucleases (Sigma Company), 1.0 μl of 10×Buffer buffer, 3 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 10 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 (Sigma Company), 2.0 μl of 10×Buffer buffer, and use ddH 2 Make up O to 20μl, and react at 16°C for 12-16h. After the ligation ...

Embodiment 3

[0042] Example 3: Denaturation, renaturation and protein purification of AlE75 fusion protein

[0043] Denaturation and renaturation of inclusion body proteins were performed according to the following steps: resuspend the bacterial cell pellet in 20ml lysis buffer (Sigma Company, 20mM Tris-HCl containing 1mM PMSF and bacteria protease inhibitorcocktail, pH 8.0), sonicate (power 400W , working for 4 sec, intermittent for 8 sec, 20 min in total); centrifuge the sonicated cell lysate at 10,000 g for 20 min at 4°C to collect the precipitate; use inclusion body washing solution (20 mM Tris, 1 mM EDTA, 2M urea, 1M NaCl, 1% Triton X- 100, pH8.0) to wash the inclusion body 3 times; dissolve the inclusion body in a certain proportion with a dissolution buffer (20mM Tris, 5mMDTT, 8M urea pH8.0), and place it at 4 degrees overnight; room temperature, 15000rpm centrifugation for 15min; Add dropwise 20mM Tris-HCl 100mM NaCl Buffer pH8.0 (Sigma Company) buffer solution, gradually double th...

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Abstract

The invention discloses a DNA sequence for coding an apolygus lucorum cell nucleus hormone receptor E75. The invention further discloses the apolygus lucorum cell nucleus hormone receptor E75, a recombinant expression vector used for coding the DNA sequence for coding the apolygus lucorum cell nucleus hormone receptor E75, and a transgenic cell system or a transgenosis recombinant bacterium, application of the recombinant expression vector, the transgenic cell system or the transgenosis recombinant bacterium in producing the apolygus lucorum cell nucleus hormone receptor E75 as well as a preparation method of the apolygus lucorum cell nucleus hormone receptor E75. A gene of the apolygus lucorum cell nucleus hormone receptor E75 is utilized to construct a pCzn-E75 recombinant plasmid; the pCzn-E75 recombinant plasmid is transferred into a TOP 10 clone strain, and can generate an inclusion body of protein after being induced through IPTG in an Arctic Express expression bacterial strain, the protein can be formed through denaturation and renaturation, and purifying is performed to prepare the protein. The prepared protein has molecular weight of about 75 KD and has protein concentration of 0.4 mg / ml.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a nuclear hormone receptor E75 of Lygus viridans, its coding sequence, carrier and bacterial strain. Background technique [0002] The green mirid bug Apolygus lucorum (Hemiptera: Miridae) belongs to the Miridae family of Hemiptera, and is an important pest on various crops such as cotton, vegetables, fruit trees, pasture and so on. In recent years, due to the large-scale planting of transgenic Bt cotton and the adjustment of the agricultural industrial structure, the population of Lygus spp. has increased sharply, and the long-term dependence on chemical pesticide control has led to the increasing resistance of Lygus spp. The prevention and control of the green Lygus is facing severe challenges, and it is urgent to develop new pollution-free prevention and control methods to reduce the use of chemical pesticides. [0003] The steroid hormone 20-hydroxyecdysone...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/72C12N15/70C12N1/21C12R1/19
CPCC07K14/721C12N15/70
Inventor 谭永安肖留斌柏立新赵静孙洋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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