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Attractant screening method based on citrus fruit fly odorant binding protein

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: 2017-08-29
四川科劲生物科技有限公司
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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 attractant formula has become a bottleneck restricting the olfactory attractants of B. dorsalis, especially the female attractants.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Collection of Bacteralis dorsalis antennal total RNA and gene cloning of odorant binding protein OBP

[0019] 1 Cloning of Bacteralis dorsalis odorant-binding protein BdorOBP2

[0020] 1.1 Extraction of Bacteralis dorsalis total RNA

[0021] The total RNA of Bactrocera dorsalis was extracted with Trizol reagent, and the specific operation steps were as follows:

[0022] 1) Take out the preserved Bactrocera dorsalis sample from -80°C, put it into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen for rapid grinding, this step is performed on ice, and weigh 100 mg after grinding into powder Put the tissue sample into a 1.5mL centrifuge tube, then add 1ml Trizol reagent, and mix on a vortex mixer;

[0023] 2) Let the sample stand at room temperature for 5 minutes to completely separate the nucleic acid-protein complex;

[0024] 3) 4°C, 12 000×g, centrifuge for 5 minutes;

[0025] 4) Transfer the supernatant to a new centrifuge tube (do not absorb ...

Embodiment 2

[0053] Example 2: Bacteralis dorsalis odorant binding protein BdorOBP2 expression vector construction

[0054] 2.1 PCR amplification of BdorOBP2 gene

[0055] The total RNA of B. dorsalis was extracted, and the first strand of cDNA obtained by reverse transcription was used as a template to carry out PCR reaction with the designed and synthesized primers before and after BdorOBP2 of B. The target fragment is consistent with the expected fragment length.

[0056] 2.2 Identification of bacterial liquid PCR

[0057] The target fragment obtained by PCR amplification of BdorOBP2 gene was recovered and purified by gel, ligated and transformed with pMD.18-T Vector, and after blue-white and ampicillin identification, the selected white clones were identified as positive clones. The PCR verification results showed that, The target stripe size meets the requirements.

[0058] 2.3 Transformation into competent cells Trans 5α

[0059] The recombinant plasmid pMD18 / BdorOBP2 was verifie...

Embodiment 3

[0074] Example 3: Induced expression of Bacteralis dorsalis odorant binding protein BdorOBP2

[0075] After verification, the single colony was cultured in 3 mL of LB medium containing 100 μg / mL ampicillin with shaking at 37 °C overnight, and inoculated into 20 mL of LB medium with a 1% (V / V) inoculation amount the next day to expand the culture to OD600 at At about 0.5, add IPTG to a final concentration of 1mmol / L and start to induce expression at 30°C and 200rpm. Take out 1 mL of bacterial liquid every 1 hour, and induce for 5 hours. Use the uninduced bacterial liquid as a negative control. After the induction, the bacterial liquid collected every hour is centrifuged at 8000rpm for 10min to precipitate, and resuspended with ddH20 and then centrifuged at 5000rpm for 10min. Collect the cells, add 150 μl of 1×SDS sample buffer to suspend the cell pellet, and boil in water at 100°C for 10 minutes to fully lyse the cells, then carry out the samples in each tube in 5% stacking gel...

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PUM

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Abstract

The invention discloses an attractant screening method based on citrus fruit fly odorant binding protein, and belongs to the technical field of biological engineering. The method comprises the following steps of collecting citrus fruit fly tentacle total RNA; obtaining the whole length of the citrus fruit fly odorant binding protein through RT-PCR; building a prokaryotic expression vector of the citrus fruit fly odorant binding protein; inducing the citrus fruit fly recombinant odorant binding protein through IPTG; performing purification through a nickel agarose gel affinity chromatographic column; obtaining a combination reaction spectrum of the citrus fruit fly recombinant odorant binding protein and the host fruit odorant volatile matter by a competitive fluorescent combination method; when the dissociation constant KD is lower than 10 mumol / L of host fruit smell, and the fluorescence competition IC50 value is smaller than 30mumol / L, determining the attractant to be the host fruit smell Attractant suitable for the citrus fruit fly. The invention provides a novel strategy for the screening and the designing of the citrus fruit fly host fruit smell odorant information attractant recipe.

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, also known as Orient fruit fly (Orient fruit fly), belongs to Diptera (Diptera), Tephritidae (Tephritidae), fruit fly genus Bactrocera Macquart, is a class of important 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 to control its harm in production, but chemical pesticides are not only easy to cause fruit and environmental pollution, but also because of their special damage mode (mainly adults lay eggs under the fruit peel, larvae live in the fruit pulp to feed, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/12C12N15/85
CPCG01N33/68C07K14/43577C12N15/85
Inventor 林丹
Owner 四川科劲生物科技有限公司
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