Cloning and functional analysis of hydroxylase gene TcCYP725A22 from Taxus wallichiana var.chinensis
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摘要: 基于中国红豆杉（Taxus wallichiana var.chinensis（Pilger） Florin）转录组序列，从中扩增到1个新的P450羟化酶基因TcCYP725A22，并对其进行了克隆、表达及功能分析。生物信息学分析结果显示：该基因编码区长1500 bp，共编码499个氨基酸；编码蛋白无信号肽，包含1个跨膜区，具有催化活性中心结构域。进一步将该基因在酿酒酵母WAT11中进行异源表达功能研究，蛋白质免疫印迹（Western blot）实验结果表明，目的蛋白TcCYP725A22可在WAT11中成功表达；液相色谱-质谱联用（LC-MS）分析结果显示，羟化酶TcCYP725A22对底物紫杉素表现出催化活性。依据质谱结果及反应底物紫杉素的分子结构，判断其催化产物可能为添加了2个羟基的紫杉烷新衍生物。Abstract: Based on our previous transcriptome analysis, a new P450 hydroxylase gene was identified and named TcCYP725A22 according to homological analysis. The TcCYP725A22 cDNA was 1500 bp in length and encoded 499 amino acids. Bioinformatics predicted that the TcCYP725A22 protein contained a transmembrane region and catalytically active domains but no signal peptide. Functional analysis of TcCYP725A22 was carried out in Saccharomyces cerevisiae strain WAT11. Western blotting results indicated that the target protein was expressed successfully in WAT11. Furthermore, LC-MS analysis showed that TcCYP725A22 expressed in yeast was able to transform taxusin to its hydroxyl derivative, indicating that it had catalytic activity.
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