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Apolygus lucorum membrane-bound trehalase, its coding sequence, vector and strain of sequence, and application of vector or strain

A technology of trehalase and chlorophyll, which is applied in the field of agricultural science, can solve the problems of lack of recombinant trehalase and no research, and achieves the effects of wide suitable reaction temperature range, speeding up reaction speed and reducing production cost.

Inactive Publication Date: 2013-09-04
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The use of insect trehalase inhibitors to control insects is a new method of pest control. However, there is a shortage of recombinant trehalase obtained from in vitro. At the same time, there is no relevant research at home and abroad on the membrane-bound trehalase of Lygus green bug.

Method used

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  • Apolygus lucorum membrane-bound trehalase, its coding sequence, vector and strain of sequence, and application of vector or strain
  • Apolygus lucorum membrane-bound trehalase, its coding sequence, vector and strain of sequence, and application of vector or strain
  • Apolygus lucorum membrane-bound trehalase, its coding sequence, vector and strain of sequence, and application of vector or strain

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Effect test

Embodiment 1

[0036] Example 1: Acquisition of ALTre-2 Gene

[0037] 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. Primers ALTre-2-F (5'-GAGTTCTACTACTGGGATTC-3') and ALTre-2-R (5'-GCGTTGGGATAGTCCCCATTG-3') suitable for PCR amplification were designed according to the conserved sequence of known insect membrane-bound trehalase genes , to obtain the conserved sequence of the membrane-bound trehalase gene of Lygus viridans, the conserved sequence is shown in SEQ ID NO: 3 in the sequence listing, the PCR reaction system is...

Embodiment 2

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

[0040] ALTre-2 gene prokaryotic expression vector construction method: The prokaryotic expression vector construction method containing ALTre-2 gene T vector is as follows: the above sequencing is connected into the pGEM-TEasy vector and pET28a is double-digested with restriction enzymes NdeI and XhoI, Perform large segment joins ( figure 2 ). 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 2Make 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 20μl, and react at 16°C for 12-16h. After the ligation product was transformed into Escherichia coli TOP10, positive clones were screened by PCR. ...

Embodiment 3

[0042] Example 3: Denaturation and renaturation of ALTre-2 fusion protein and purification of protein

[0043] Denaturation of inclusion body protein was carried out according to the following steps: centrifuge at 4000g, 4°C for 15min, discard the supernatant, wash the bacteria twice with PBS buffer, resuspend in PBS buffer, break the cells under high pressure, centrifuge at 12000g, 4°C for 30min, and save the precipitate For inclusion body, add 60mL binding buffer (6M guanidine hydrochloride, 100mM Tris, 300mM NaCl, pH8.0), resuspend inclusion body, stir to dissolve, centrifuge at 16000g, 4℃ for 30min, keep the supernatant; Elution (buffer A: 6M guanidine hydrochloride, 100mM Tris, 300mM NaCl, pH8.0; buffer B: 8M urea, 100mM Tris, 300mM NaCl, pH8.0; buffer C: 8M urea, 100mM Tris, 300mM NaCl, different concentrations of imidazole, pH8.0); then use 10 times the volume of the column bed Binding buffer (20mM Tris-HCl pH7.9, 10mM imidazole, 0.5M NaCl) to wash the equilibrated colu...

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Abstract

The invention discloses an Apolygus lucorum membrane-bound trehalase coding DNA sequence. The invention also discloses an Apolygus lucorum membrane-bound trehalase, a recombinant expression vector or a transgenic cell system or a transgenic transgenic bacterium of the Apolygus lucorum membrane-bound trehalase coding DNA sequence, an application of the recombinant expression vector or the transgenic cell system or the transgenic transgenic bacterium in the production of the Apolygus lucorum membrane-bound trehalase, and a preparation method of the Apolygus lucorum membrane-bound trehalase. The molecular weight of zymoprotein prepared in the invention is 60KD, and zymoprotein can degrade trehalose to form glucose at an optimal temperature of 50DEG C under an optimal pH value of 7.0. Purified trehalase can be used for the qualitative and quantitative content detection of trehalase in industries and other fields, and provides a foundation for the research and development of insect trehalase inhibitors.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a membrane-bound trehalase from Lygus viridans, its coding sequence, carrier, bacterial strain and application. 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] Membrane-bound trehalase (A...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/63C12N5/10C12N1/21C12N15/70C12R1/19
Inventor 谭永安肖留斌柏立新孙洋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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