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黄烷酮3-羟化酶(flavanone 3-hydroxylase,F3H)是类黄酮代谢途径中的关键酶。本研究采用逆转录PCR技术,获取了茶树(Camellia sinensis)F3H的开放阅读框(open reading frame,ORF)包含1 107个碱基,编码含有368个氨基酸的蛋白质,推测分子量为41.46 kD,等电点为5.61;实时荧光定量PCR表明,CsF3H在叶片中表达量较高,且受光照影响;将此基因重组到表达载体PRSF上,导入大肠杆菌(Escherichia coli)BL21中进行原核表达,优化原核表达的条件,结果表明,最佳诱导温度28℃、异丙基-β-d-硫代半乳糖苷(IPTG)浓度1.0 mmol/L、诱导时间5 h;利用高效液相色谱法(HPLC)对重组蛋白进行了体外酶活的检测,结果表明,重组目的蛋白具有F3H酶活性,可将反应底物柚皮素(N)和圣草酚(E)分别转化为二氢山奈素(DHK)和二氢槲皮素(DHQ);当E作为底物时,酶活性明显高于柚皮素。本研究结果为茶树F3H酶动力学研究和其器官组织特异性研究提供了基础资料。
Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid metabolism pathway. In this study, the open reading frame (ORF) of Camellia sinensis F3H was obtained by reverse transcription PCR. It contained 1 107 bases and encoded a protein with 368 amino acids. The deduced molecular weight was 41.46 kD. Point was 5.61. Real-time fluorescence quantitative PCR showed that CsF3H was highly expressed in leaves and affected by light. The gene was recombined into expression vector PRSF and introduced into Escherichia coli BL21 for prokaryotic expression optimization The results showed that the optimum induction temperature was 28 ℃, the concentration of isopropyl-β-d-thiogalactoside (IPTG) was 1.0 mmol / L and the induction time was 5 h. The recombinant protein was tested for in vitro enzymatic activity. The results showed that the recombinant protein had the activity of F3H, and the substrate of naringenin (N) and eriodictyol (E) were transformed into dihydrokadalactin (DHK) and Dihydroquercetin (DHQ); When E as a substrate, the enzyme activity was significantly higher than naringenin. The results of this study provide the basic information for the enzyme kinetics of F3H and its organ tissue specificity.