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Issue:ISSN 1000-7083
          CN 51-1193/Q
Director:Sichuan Association for Science and Technology
Sponsored by:Sichuan Society of Zoologists; Chengdu Giant Panda Breeding Research Foundation; Sichuan Association of Wildlife Conservation; Sichuan University
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Your Position :Home->Past Journals Catalog->2019 Vol.38 No.2

cDNA Cloning, Prokaryotic Expression, and Enzyme Activity Analyses of far3 Gene from Ericerus pela
Author of the article:ZHAO Zunling, WANG Xueqing, YANG Pu*
Author's Workplace:Research Institute of Resource Insects, Chinese Academy of Forestry, Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Kunming 650224, China
Key Words:Ericerus pela; fatty acyl-CoA reductase; rapid amplification of cDNA ends; prokaryotic expression; enzyme activity analyses
Abstract:Fatty acyl-CoA reductases (FARs) reduce fatty acyl-CoA to corresponding fatty alcohols, and therefore play a key role in the biosynthesis of wax ester. In this study, the full length cDNA of far3 gene in Ericerus pela was obtained by rapid amplification of cDNA ends. The open reading frame (ORF) was found to be 1 566 bp. Phylogenetic analysis showed that, FAR3 of E. pela was clustered together with those of Homo sapiens, Mus musculus, and Drosophila melanogaster. The recombinant plasmid of pET-30a eGFP/EpelFAR3 was successfully constructed and then transferred into Escherichia coli BL21 competent cells for prokaryotic expression. The protein was highly expressed when the isopropyl thiogalactoside concentration was 0.05 mmol·L-1 and the induction time was 6 hours. Western blot validated the molecular weight of the expressed protein was consistent with theoretical prediction. The result of LC-MS-MS analysis confirmed the expression of FAR3 protein based on protein score (3 900) and coverage (74%). The enzyme activity of FAR3 was tested with different substrates, which included C24, C26, C28, and C30 fatty acyl-CoA. The reaction products were further detected using gas chromatography, and no corresponding fatty alcohol was identified. The ORF sequence of far3 gene and protein expression data are of considerable importance for further functional and histological location studies.
2019,38(2): 149-156 收稿日期:2018-11-08
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