茶树二氢黄酮醇4-还原酶基因的克隆、表达及功能分析

摘要

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of catechins in tea plant. However, the functions and the zymologic properties of DFR were not deeply identified in recent researches. The open reading frame of DFR gene, which encoding a 347 amino acids protein, was cloned from tea plant (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 38.69 kD and its theoretical isoelectric point was 6.02. The gene was cloned into the expression vector SUMO for expression in prokaryotic cells. The SDS-PAGE results showed that the dihydroflavonol 4-reductase peoteins was expressed in Escherichia coli BL21. The optimal inducing conditions including time, temperature and IPTG concentration were studied. The deduced protein was purified and its activity was detected by HPLC-MS method. The results indicated that purified protein showed the DFR activity, catalyzed the reduction reaction of DHQ and DHM. The research provides a valuable foundation for better understanding the substrate specificity and enzymatic properties of CsDFR.%　　茶树二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase, DFR）是儿茶素合成途径中的关键酶。本研究采用 RT-PCR 技术，获得了茶树二氢黄酮醇4-还原酶基因（CsDFR）的开放阅读框，它编码含347个氨基酸的蛋白质，推测分子量为38.69 kD，等电点为6.02。成功地将该基因重组到表达载体 SUMO 上，并在大肠杆菌 BL21中进行原核表达；优化了原核表达中诱导时间、诱导温度、IPTG 浓度；纯化出目的蛋白。利用 HPLC-MS 方法对重组蛋白进行了体外酶活检测，结果表明目的蛋白具有 DFR 酶活性，可催化 DHQ 和 DHM 的还原反应。