铁线蕨中氧甲基转移酶基因克隆与功能鉴定

doi:10.6040/j.issn.1671-7554.0.2019.027

摘要/Abstract

摘要： 目的克隆并鉴定铁线蕨中参与黄酮及苯丙类化合物生物合成的氧甲基转移酶(OMTs)。方法以铁线蕨的叶片总RNA为模板,采用RT-PCR技术,从中获得两个OMTs cDNA全长,分别命名为AcOMT1和AcOMT2,并将其构建到蛋白表达载体pET32a中,在大肠杆菌BL21中进行蛋白表达,利用镍离子亲和层析柱纯化重组蛋白。在合适的反应条件下进行体外酶活分析。利用Swiss-model在线服务器分别生成AcOMT1和AcOMT2三维同源模型,通过PyMOL软件预测影响蛋白活性的关键氨基酸位点。利用MEGA v5.1和DNAMAN 7.0.2软件分别进行系统发育进化树分析和氨基酸多序列比对。结果从铁线蕨的转录组测序数据库中筛选并克隆得到两个OMTs,序列比对与进化树分析结果表明它们均属于Ⅰ型OMTs。酶活结果显示AcOMT1和AcOMT2均可催化多种底物,催化特点相似,其最适底物均为槲皮素。结论从铁线蕨中克隆并鉴定了两个OMT基因,对阐明蕨类植物中黄酮类化合物的甲基化修饰具有一定的理论指导意义。

关键词: 铁线蕨, 黄酮类化合物, 氧甲基转移酶, 基因克隆, 功能鉴定

Abstract: Objective To isolate and characterize the O-methyltransferase genes involved in the biosynthesis of flavonoids and phenypropanoids from Adiantum capillus-veneris L. Methods The full-length cDNA sequences of two O-methyltransferase genes were cloned by the RT-PCR method using the total RNA isolated from the Adiantum capillus-veneris L. as template, and designated as AcOMT1 and AcOMT2, respectively. The amplified ORFs were ligated into the pET-32a vector, and then were transformed into E.coli BL21 for expression. The recombinant proteins were purified using the Ni-NTA Sefinose His-bind column and used for enzyme assays. The protein homology models of AcOMT1 and AcOMT2 were generated by Swiss-model on-line server, and the key amino acids which were postulated to affect protein activity were predicted by PyMOL software. The analyses of phylogenetic tree and multiple sequence alignment were performed using MEGA v5.1 and DNAMAN 7.0.2 software, respectively. Results Two genes encoding putative O-methyltransferase were identified from the transcriptome sequencing database of Adiantum capillus-veneris L. and cloned from the plants. The sequence alignment and phylogenetic analysis indicated that AcOMTs belong to the family of type Ⅰ OMTs. Meanwhile, the enzymatic assays showed that AcOMT1 and AcOMT2 had the similar catalytic characteristics and broad substrate selectivety. Quercetin was the favorite substrate. Conclusion Two OMT genes were isolated and characterized from Adiantum capillus-veneris L., which would provide theoretical significance to elucidate the methylation modification of flavonoids in ferns.

Key words: Adiantum capillus-veneris L., Flavonoids, O-methyltransferase, Gene cloning, Functional characterization

中图分类号:

Q789

倪荣,张晓双,程爱霞. 铁线蕨中氧甲基转移酶基因克隆与功能鉴定[J]. 山东大学学报 (医学版), 2019, 57(4): 27-33.

NI Rong, ZHANG Xiaoshuang, CHENG Aixia. Cloning and functional characterization of two O-methyltransferases from Adiantum capillus-veneris L.[J]. Journal of Shandong University (Health Sciences), 2019, 57(4): 27-33.

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