白蜡虫far3基因cDNA全长克隆、原核表达和酶活分析

脂酰辅酶A还原酶(FAR)可将脂酰辅酶A还原为相应的脂肪醇,在白蜡生物合成中起至关重要的作用。本研究通过cDNA末端快速扩增技术获得白蜡虫Ericerus pela far3基因cDNA全长,其开放阅读框(ORF)1 566 bp。对白蜡虫FAR3编码蛋白进行系统发育分析,发现FAR3与人类Homo sapiens、小鼠Mus musculus、黑腹果蝇Drosophila melanogaster等物种的FAR聚为一支;成功构建pET-30a eGFP/EpelFAR3原核表达质粒,转入大肠杆菌Escherichia coli BL21感受态细胞,在浓度为0.05 mmol·L^-1的异丙基硫代半乳糖苷诱导6 h后有较高的蛋白表达量;经Western blot验证,表达蛋白分子量与预估蛋白分子量符合;质谱分析蛋白质分值为3 900,肽段覆盖度74%,所得肽段与理论序列相符;利用底物C24脂酰辅酶A、C26脂酰辅酶A、C28脂酰辅酶A和C30脂酰辅酶A对原核表达蛋白进行活性分析,利用气相色谱进行蛋白活性验证,没有理论产物相应脂肪醇的生成。本研究中白蜡虫far3 cDNA ORF的获得及原核表达的实现,为进一步的功能和组织表达定位研究奠定了基础。

cDNA Cloning,Prokaryotic Expression,and Enzyme Activity Analyses of far3 Gene from Ericerus pela

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.