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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。
Dihydroflavonol 4-reductase (DFR) is a key enzyme in the catechin synthesis pathway. In this study, RT-PCR technology was used to obtain the open reading frame of the tea 4-reductase gene (CsDFR), which encodes a protein of 347 amino acids with a predicted molecular weight of 38.69 kD and an isoelectric point of 6.02. The gene was successfully recombined into the expression vector SUMO and expressed in E. coli BL21 in prokaryotic expression. The induction time, induction temperature and IPTG concentration in prokaryotic expression were optimized, and the target protein was purified. The recombinant protein was detected by HPLC-MS in vitro. The results showed that the target protein had DFR activity and could catalyze the reduction of DHQ and DHM.