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Smell activator screening method based on apis cerana odorant binding protein

An odor-binding protein and technology of Apis cerana, applied in the field of bioengineering, can solve the problems of various and complex volatile components

Inactive Publication Date: 2017-06-13
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the variety and complexity of the volatile components of various plant and floral fragrances, how to quickly screen out a suitable olfactory active agent formula has become a bottleneck restricting the development and design of olfactory active agents in Apis cerana.

Method used

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  • Smell activator screening method based on apis cerana odorant binding protein
  • Smell activator screening method based on apis cerana odorant binding protein
  • Smell activator screening method based on apis cerana odorant binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Collection of total RNA from antennae of Apis cerana and gene cloning of odorant binding protein OBP

[0029] 1 Cloning of the odorant-binding protein AcerOBP2 of Apis mellifera

[0030] 1.1 Extraction of total RNA from Apis cerana

[0031] Trizol reagent was used to extract the total RNA of the antennae of Apis cerana workers, and the specific operation steps were as follows:

[0032] 1) Take out the preserved antennae samples of Apis chinensis worker bees from -80°C, put them into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen to quickly grind, this step is operated on ice, and weigh 100mg 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;

[0033] 2) Let the sample stand at room temperature for 5 minutes to completely separate the nucleic acid-protein complex;

[0034] 3) 4°C, 12 000×g, centrifuge for 5 minutes;

[0035] 4) Transfer the supernatant to a n...

Embodiment 2

[0068] Example 2: Construction of the expression vector for the odorant binding protein AcerOBP2 of Apis mellifera

[0069] 2.1 PCR amplification of AcerOBP2 gene

[0070] The total RNA of Apis chinensis was extracted, the first strand of cDNA obtained by reverse transcription was used as a template, and the primers before and after primers of AcerOBP2 were designed and synthesized for PCR reaction, and the PCR results were subjected to agarose gel electrophoresis, as shown in figure 1 As shown in , the target fragment of about 447bp was obtained, which was consistent with the expected fragment length.

[0071] 2.2 Identification by double enzyme digestion

[0072] The target fragment obtained by PCR amplification of AcerOBP2 gene was recovered and purified by gel, ligated with pGEM-T easy Vector for transformation, and after identification of blue and white spots, the white clone was selected on the ampicillin-containing medium plate, inoculated and cultured, and the plasmid...

Embodiment 3

[0092] Example 3: Induced Expression of Apis mellifera Odor Binding Protein AcerOBP2

[0093] After verification, the single colony was cultured in 3 mL LB medium containing 100 μg / mL ampicillin with shaking at 37 ° C overnight, and inoculated into 20 mL LB medium with 1% (V / V) inoculum the next day to expand the culture to OD 600 At about 0.4, add IPTG to a final concentration of 1 mmol / L and start to induce expression at 30°C and 200 rpm. Take out 1mL of bacterial liquid every 1h, induce 5h in total, use the uninduced bacterial liquid as a negative control, after the end of induction, centrifuge the bacterial liquid collected every hour at 8000rpm for 10min, and use ddH 2 O were resuspended and centrifuged at 5000rpm for 10 minutes to collect the bacteria, add 150 μl of 1×SDS sample buffer to suspend the bacteria pellet, and boil in water at 100°C for 10 minutes to fully lyse the bacteria. 5% stacking gel and 12% separating gel were electrophoresed in SDS-PAGE to determine ...

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Abstract

The invention discloses a plant fragrance smell activator formula screening method based on apis cerana odorant binding protein. The method comprises the following process steps: 1) extracting working bee antenna total RNA (Ribonucleic Acid) of apis cerana; 2) designing an apis cerana odorant binding protein gene primer, and acquiring the full length of an apis cerana odorant binding protein gene through RT-PCR (Reverse Transcription-Polymerase Chain Reaction); 3) building a prokaryotic expression vector of the apis cerana odorant binding protein gene; 4) inducing recombinant expression of the apis cerana odorant binding protein through IPTG (isopropyl-beta-d-thiogalactoside), and performing purification through a Ni Sepharose FF affinity column; 5) acquiring a combined response spectrum of the apis cerana odorant binding protein and plant fragrance smell through a competitive fluorescent combination method, and determining the combined response spectrum as the plant fragrance smell activator suitable for the apis cerana when the dissociation constant KD is lower than 20mummol / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a formula screening method for a plant floral scent olfactory active agent based on the scent binding protein of Apis chinensis. Background technique [0002] Chinese honey bee (Apis cerana cerana, referred to as "Apis cerana") is an excellent bee species native to China, and it is also the head bee species of beekeepers in China for thousands of years. Ecosystems such as Brassicaceae, Rosaceae, Anacardiaceae and Camellia such as plant floral fragrances produce good olfactory sensitivity and adaptability. In recent years, with the development of facility agriculture, especially in the process of strawberry facility agriculture production in winter, the use of bee pollination has become an indispensable production process to ensure high yield and high quality of strawberry. However, in the pollination process of using bees at present, the pollination efficiency...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/435C07K1/22G01N21/64
CPCC07K14/43572C12N15/70C12N2800/101G01N21/64G01N21/6486G01N2021/6421
Inventor 李红亮张林雅吴帆
Owner CHINA JILIANG UNIV
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