Construction and Functions of Expression Vector pIB/V5-His of <i>AcerOr1</i> in <i>Apis cerana</i>

GUO Li-na; ZHAO Hui-ting; REN You-she; XU Bing; JIANG Yu-suo

doi:10.19303/j.issn.1008-0384.2020.04.001

Volume 35 Issue 4

Apr. 2020

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Article Navigation > Fujian Journal of Agricultural Sciences > 2020 > 35(4): 359-365

GUO L N, ZHAO H T, REN Y S, et al. Construction and Functions of Expression Vector pIB/V5-His of AcerOr1 in Apis cerana [J]. Fujian Journal of Agricultural Sciences，2020，35（4）：359−365 doi: 10.19303/j.issn.1008-0384.2020.04.001

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GUO L N, ZHAO H T, REN Y S, et al. Construction and Functions of Expression Vector pIB/V5-His of AcerOr1 in Apis cerana [J]. Fujian Journal of Agricultural Sciences，2020，35（4）：359−365 doi: 10.19303/j.issn.1008-0384.2020.04.001

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Construction and Functions of Expression Vector pIB/V5-His of AcerOr1 in Apis cerana

doi: 10.19303/j.issn.1008-0384.2020.04.001

1.
College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi　030801, China
2.
College of Life Science, Shanxi Agricultural University, Taigu, Shanxi　030801, China
3.
College of Engineering, Shanxi Agricultural University, Taigu, Shanxi　030801, China

Received Date: 2019-12-24
Rev Recd Date: 2020-02-23
Publish Date: 2020-04-01

Abstract

Abstract

Objective To construct and determine the functions of a recombinant expression vector which could stably and efficiently express the olfactory receptor AcerOr1 in Sf9 cells of Apis cerana. Method AcerOr1 was amplified and using T4 DNA ligase ligated into the insect expression vector pIB/V5-His treated by BamHI and EcoRI. The recombinant DNA was liposome-transfected onto Sf9. Subsequently, AcerOr1 expression was determined by Western blot, and protein subcellular localization by immunofluorescence. A full-wavelength multifunctional microplate reader was employed to determine the intracellular Ca²⁺ concentration before and after the recombinant receptor was stimulated separately with lauric acid, linolenic acid, α–terpineol, and undecanoic acid. Result The recombinant pIB/V5-AcerOr1 was successfully constructed with a stable, transfected vector expression. The recombinant expression vector was determined to be approximately 50 kDa and localized as predicted in the Sf9 cell membrane. The intracellular Ca²⁺ concentrations in the Sf9 cells transfected with pIB/V5-AcerOr1 increased correspondingly upon the stimulations by the floral substances. Conclusion The recombinant pIB/V5-AcerOr1 was successfully constructed with a stable expression in the Sf9 cell of the bees with the olfactory function verified by exposing the cells to aromatic chemicals.
- Apis cerana,
- olfactory receptor AcerOr1,
- pIB/V5-His,
- expression,
- subcellular localization,
- function analysis

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References(23)

References

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