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          CN 51-1193/Q
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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
分类号:Q78
基金项目:中央级公益性科研院所基本科研业务费专项(CAFYBB2017ZB005);林业公益性行业科研专项(201504302);国家自然科学基金项目(31572337)
作者简介:赵遵岭(1993-),女,硕士研究生,研究方向:昆虫分子生物学,E-mail:zhaozl_blue@163.com
*通信作者:杨璞,研究员,主要从事昆虫分子生物学研究,E-mail:zjuyangpu@aliyun.com
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