一个蛇苔氧甲基转移酶基因的克隆与功能鉴定

目的 克隆蛇苔氧甲基转移酶(CcOMT1)基因并对其进行功能鉴定。 方法 通过RT-PCR技术获得CcOMT1基因的cDNA全长。构建原核表达质粒pET32a-CcOMT1,在大肠杆菌BL21(DE3)中表达后,利用Ni-NTA吸附柱对表达的重组蛋白进行纯化,并通过体外酶活分析其活性。采用DNAMAN 7.0和MEGA 5.1软件分别进行氨基酸多序列比对和进化关系分析。 结果 克隆得到的CcOMT1开放读码框为837 bp,编码278个氨基酸,预测分子量为30.95 kDa,等电点为5.59。与来自钝鳞紫背苔、紫花苜蓿和冰叶日中花的氧甲基转移酶的同源性分别为73.18%、53.60%和44.60%。对纯化的重组蛋白进行酶活检测,结果表明融合蛋白可以催化具有邻二酚羟基的黄酮和苯丙类化合物,产生相应的氧甲基化产物,其最适底物为槲皮素。 结论 克隆并鉴定了一个CcOMT1基因,为通过酶法实现化合物的氧甲基化修饰提供了一个候选基因。     

Terpenoids and bisbibenzyls from Chinese liverworts Conocephalum conicum and Dumortiera hirsuta

Four new monoterpene esters, 2α,5β-dihydroxybornane-2-cinnamate (1), 2α,5β-dihydroxybornane-5-acetyl-2-cinnamate (2), 2α,5β-dihydroxybornane-2-p-hydroxycinnamate (3) and 2α,5β-dihydroxy-bornane-2-cis-p-hydroxycinnamate (4), together with a known compound 3,4-dimethoxystyrene (5) were isolated from Chinese liverwort Conocephalum conicum and six known compounds, 5,7-dihydroxycalamenene (6), 7-hydroxycalamenene (7), lunularin (8), riccardin C (9), marchantin C (10) and riccardin D (11) were isolated from Dumortiera hirsuta. Their structures were elucidated by extensive spectral analysis and chemical correlations. Compounds 1 and 8 showed moderate cytotoxicity against human HepG2 cells with IC₅₀ 4.5 μg/ml and 7.4 μg/ml, respectively, while compound 8 also showed antimicrobacterial activity against Pseudomonas aeruginosa with minimum inhibitory concentration at 64 μg/ml.