Citrus fruit fly odorant binding protein-based attractant screening method

An odor-binding protein and Bactrocera dorsalis technology, applied in the field of bioengineering, can solve problems such as complex volatile components

Inactive Publication Date: 2013-08-14
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of the volatile components of various fruits, how to screen out a suitable attractan...

Method used

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  • Citrus fruit fly odorant binding protein-based attractant screening method
  • Citrus fruit fly odorant binding protein-based attractant screening method
  • Citrus fruit fly odorant binding protein-based attractant screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Collection of Bacteralis dorsalis antennal total RNA and gene cloning of odorant binding protein OBP

[0027] 1 Cloning of Bacteralis dorsalis odorant-binding protein BdorOBP2

[0028] 1.1 Extraction of Bacteralis dorsalis total RNA

[0029] The total RNA of Bactrocera dorsalis was extracted with Trizol reagent, and the specific operation steps were as follows:

[0030] 1) Take out the preserved Bactrocera dorsalis sample from -80 ℃, put it into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen to grind quickly, this step is performed on ice, and after being ground into powder, weigh 100 mg tissue sample into a 1.5 mL centrifuge tube, then add 1 ml Trizol reagent, and mix on a vortex mixer;

[0031] 2) Let the sample stand at room temperature for 5 minutes to completely separate the nucleic acid-protein complex;

[0032] 3) Centrifuge at 12 000×g for 5 minutes at 4°C;

[0033] 4) Transfer the supernatant to a new centrifuge tube (do not suck t...

Embodiment 2

[0064] Example 2: Bacteralis dorsalis odorant binding protein BdorOBP2 expression vector construction

[0065] 2.1 PCR amplification of BdorOBP2 gene

[0066] The total RNA of Bactrocera dorsalis was extracted, the first strand of cDNA obtained by reverse transcription was used as a template, and the front and back primers of BdorOBP2 designed and synthesized were used for PCR reaction, and the PCR results were electrophoresed on agarose gel, as shown in figure 1 As shown in , the target fragment of about 447bp was obtained, which was consistent with the expected fragment length.

[0067] 2.2 Identification of bacterial liquid PCR

[0068] The target fragment obtained by PCR amplification of the BdorOBP2 gene was recovered and purified by gel, connected with pMD?18-T Vector and transformed, after blue-white and ampicillin identification, the selected white clone was identified as a positive clone, and the PCR verification results were as follows figure 2 As shown, the targ...

Embodiment 3

[0088] Example 3: Induced expression of Bacteralis dorsalis odorant binding protein BdorOBP2

[0089] After verification, the single colonies were cultured in 3 mL LB medium containing 100 μg / mL ampicillin with shaking at 37°C overnight, and inoculated into 20 mL LB medium at an inoculation volume of 1% (V / V) for expansion on the next day When the OD600 is around 0.5, add IPTG to a final concentration of 1 mmol / L and start to induce expression at 30°C and 200 rpm. Take out 1 mL of bacterial liquid every 1 h, induce 5 h in total, and use uninduced bacterial liquid as a negative control. After the induction, the bacterial liquid collected every hour is centrifuged at 8000 rpm for 10 min to precipitate, and washed with ddH 2 0 were resuspended and centrifuged at 5000rpm for 10 minutes to collect the bacteria, and 150 μl of 1×SDS sample buffer was added to suspend the bacteria pellet, and boiled at 100°C for 10 minutes to fully lyse the bacteria, and then the samples in each tube ...

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Abstract

The invention discloses a citrus fruit fly odorant binding protein-based attractant screening method belonging to the technical field of bioengineering. The citrus fruit fly odorant binding protein-based attractant screening method comprises the following steps of: collecting the total RNA (Ribonucleic Acid) of antennae of a citrus fruit fly; obtaining the overall length of a citrus fruit fly odorant binding protein through RT-PCR (Reverse Transcription-Polymerase Chain Reaction); constructing a prokaryotic expression vector of the citrus fruit fly odorant binding protein; inducing the expression of a citrus fruit fly recombinant odorant binding protein through IPTG (isopropyl-beta-d-thiogalactoside), and purifying the citrus fruit fly recombinant odorant binding protein through a nickel sepharose gel affinity column; obtaining a conjugation reaction spectrum of the citrus fruit fly recombinant odorant binding protein and a host fruit odor volatile matter through a competitive fluorescence combing method, wherein a dissociation constant (KD) is lower than below 10 mu mol/L host fruit smell; and the IC 50 value of fluorescence competition is less than 30 mu mol/L, determining a host fruit smell attractant suitable for the citrus fruit fly. The invention provides a new strategy for screening and designing a citrus fruit fly odorant host fruit smell odor information attractant formula.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an attractant screening method based on Bactrocera dorsalis odorant binding protein. Background technique [0002] Bactrocera dorsalis Bactrocera dorsalis , also known as Orient fruit fly (Orient fruit fly), belongs to Diptera (Diptera), Tephritidae (Tephritidae), fruit fly genus Bactrocera Macquart , is an important class of quarantine pests. The insect has a wide range of hosts, and it is reported that it can damage more than 250 kinds of fruits in 46 families. It is seriously harmful to bananas, guavas, mangoes and citrus, and has caused huge economic losses to the fruit industry in my country. At present, chemical control is mainly used in production to control its hazards, but chemical pesticides are not only likely to cause fruit and environmental pollution, but also because of their special damage methods (mainly adults lay eggs under the fruit peel, larvae live ...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12N15/12C12N15/70C07K14/435C07K1/22
Inventor 李红亮陈玲商晗武王强
Owner CHINA JILIANG UNIV
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