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Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof

A technology of green ligus and air valves, applied in the field of agricultural science, can solve problems such as the rise of green ligus

Inactive Publication Date: 2014-05-14
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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  • Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof
  • Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof
  • Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the acquisition of ALUSP gene

[0032] 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, 2 μl 10 mm old NTP, 1 μl ribonucleotide inhibitor, 2 μl reverse transcriptase, 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. The primers ALUSP-F (5'-CATTATGGGWGTYTAYAGYTGTG-3') and ALUSP-R (5'-AGHAGTTCATTCCAVCCTGCTC-3') suitable for PCR amplification were designed according to the conserved sequence of the known insect hyperventilation protein to obtain the hyperventilation protein of the green Lygus bug Conserved sequence, see SEQID: 3 in the sequenc...

Embodiment 2

[0034] The construction of embodiment 2 ALUSP gene prokaryotic expression vector

[0035]The method of constructing the prokaryotic expression vector containing the ALUSP gene T vector is as follows: the target gene and the pEGX-6P-1 vector that are connected to the above sequencing into the pMD18-T vector are both digested with restriction endonucleases EcoRI and XhoI, and the large fragments are ligated ( image 3 ). 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 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. Trans...

Embodiment 3

[0037] Example 3: Denaturation and renaturation of ALUSP fusion protein and purification of protein

[0038] Denaturation of inclusion body protein was performed according to the following steps: centrifuge at 4000g, 4°C for 15min, discard the supernatant, wash the bacteria twice with PBS, resuspend in PBS, break the cells under high pressure, centrifuge at 12000g, 4°C for 30min, retain the precipitated inclusion body, Add 60ml binding buffer (7mol L -1 Guanidine hydrochloride, 50mmol·L-1Tris, 2MmDTT, 0.1%Triton-100, pH8.0), resuspend the inclusion body, stir to dissolve, centrifuge at 16000g, 4℃ for 30min, keep the supernatant; then use 10 times the volume of the column bed Bindingbuffer to wash and equilibrate the column, the flow rate is 60ml h -1 ; Then put the sample (solution supernatant) on the column at a flow rate of 45ml h-1, collect the permeate, and wash the column with Bindingbuffer 10 times the volume of the column bed at a flow rate of 60ml h -1 ; Finally use ...

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Abstract

The invention discloses a DNA (Deoxyribonucleic Acid) sequence for coding an ultraspiracle protein of a green plant bug. The invention further discloses the ultraspiracle protein of the green plant bug, a recombinant expression vector, a transgenic cell system or a transgenic recombinant bacterium of the DNA sequence for coding the ultraspiracle protein of the green plant bug, application of the recombinant expression vector, the transgenic cell system or the transgenic recombinant bacterium in production of the ultraspiracle protein of the green plant bug as well as a preparation method of the ultraspiracle protein of the green plant bug. The protein is prepared by the following steps: constructing a pEGX-6P-1 prokaryotic expression vector by means of the ultraspiracle protein gene of the green plant bug; generating an inclusion body of the protein through IPTG (Isopropyl-beta-D-Thiogalactoside) induction in escherichia coli Rostetta gami B; then, denaturing and renaturing to form the protein and purifying the protein. The molecular weight of the prepared protein is about 65KD, and the protein concentration is 1.67mg / ml. The purified ultraspiracle protein can provide a previous theoretical basis for a novel insecticide taking an ecdysone receptor as a target.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a Lygus viridans supervalve protein, its coding sequence, carrier and bacterial strain. Background technique [0002] 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, pastures, etc. 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 (20-hydroxyecdysone, 20E) is the mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/435
Inventor 谭永安肖留斌柏立新孙洋赵静
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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