中文摘要：

黄烷酮3-羟化酶（flavanone3-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（Camelliasinensis）F3H的开放阅读框（openreadingframe，omq包含1107个碱基，编码含有368个氨基酸的蛋白质，推测分子量为41．46kD，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（Escherich—iacoli）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1．0mmoFL、诱导时间5h；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m）和圣草酚（E）分别转化为二氢山奈素（DHKl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。

英文摘要：

Flavanone 3-hydroxylase （F3H） is a key enzyme in the metabolic pathways of flavonoid com- pounds in plants. The open reading frame（ORF） of F3H gene, which included 1 107 basic group and encoded a 368 amino acids protein, was cloned from tea （Camellia sinensis） by RT-PCR. The deduced protein molecu- lar weight was 41.46 kD and its theoretical isoelectric point was 5.61. The results of Real-time fluorescent quantitative PCR showed that CsF3H had high expression levels in the leaves, and was influenced by light condition. The gene was cloned into the expression vector PRSF for expression in prokaryotic cells. The SDS- PAGE results showed that the F3H peotein was expressed in Escherichia coli BL21. The optimal inducing con- ditions including temperature, isopropyl-13-d-thiogalactoside（IPTG） concentration and time were studied, and were 28 ＊C, 1.0 mmol/L, 5 h, respectively. The activity of the recombinant protein in vitro was tested by high performance liquid chromatography（HPLC） method. Experiment results showed that purified protein had the F3H enzyme activity and transfered naringenin（N） and eriodictyol（E） respectively to dihydrokaempfero （DHK） and dihydroquercetin（DHQ）, while the recombinant protein had a much more preference for eriodic- tyol（E）. The research provides basic data for the kinetic study and organ specificity of CsF3H protein.