滇重楼糖基转移酶基因的克隆和原核表达  

Cloning and Prokaryotic Expression of Glycosyltransferase Genes from Paris polyphylla var. yunnanensis

作　　者：郭思远[1] 尹艳[1,2] 石颖慧 李佳 高伟[1,3] 张夏楠[1] GUO Si-yuan;YIN Yan;SHI Ying-hui;LI Jia;GAO Wei;ZHANG Xia-nan(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;School of Pharmaceutical Sciences,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]北京中医药大学中药学院,北京102488 [3]首都医科大学药学院,北京100069

出　　处：《中国实验方剂学杂志》2021年第8期126-134,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中央本级重大增减支项目(2060302-1806-03);国家自然科学基金面上项目(81974515);北京市自然科学基金面上项目(7182018)。

摘　　要：目的:克隆滇重楼皂苷类成分生物合成途径中关键酶糖基转移酶基因(PpUGT1,PpUGT7),并对其进行生物信息学分析、相对表达量分析和原核表达。方法:试剂盒法提取滇重楼RNA并反转录,根据滇重楼转录组数据设计特异性引物,克隆基因全长,使用软件对其进行生物信息学分析,利用实时荧光定量聚合酶链式反应(Real-time PCR)检测基因相对表达量,构建原核表达载体并在大肠埃希菌中诱导表达目的蛋白。结果:PpUGT1和PpUGT7分别长1 827 bp和1 380 bp,分别编码608和459个氨基酸,相对分子质量分别为67.6 kDa和51.3 kDa,其中PpUGT1预测为甾体类糖基转移酶,PpUGT7预测为三萜类糖基转移酶。两蛋白均为亲水性蛋白,无跨膜结构,无信号肽,均与同类蛋白具有较高的保守性。实时荧光定量聚合酶链式反应(Real-time PCR)结果显示,PpUGT1的表达量由高到低依次为根>叶>花>茎,PpUGT7的表达量由高到低依次为茎>叶>花>根。此外,成功在大肠埃希菌中以可溶形式表达目的蛋白。结论:克隆得到PpUGT1和PpUGT7基因,证明其在不同植物器官中存在差异表达,并在大肠埃希菌中成功表达其重组蛋白,为进一步鉴定PpUGTs功能、解析滇重楼中皂苷类成分生物合成途径奠定基础。Objective:To clone the full-length glycosyltransferase genes(PpUGT1,PpUGT7)related to saponins biosynthesis in Paris polyphylla var. yunnanensis,and perform bioinformatics analysis,relative expression analysis and prokaryotic expression analysis. Method: Total RNA was isolated from P. polyphylla var. yunnanensis with use of the Eastep? Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)and the prokaryotic expression vectors were built to heterologously express recombinant protein in Escherichia coli. Result: The open reading frame(ORF) of PpUGT1 was 1 827 bp,encoding 608 amino acids,and was predicted as a steroid glycosyltransferase;the ORF of PpUGT7 was 1 380 bp,encoding 459 amino acids,and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively,and both of them were hydrophilic proteins,no transmembrane domain,no signal peptides,both showing high similarity and conservativeness with homologous sequences. The results of Realtime PCR showed that the expression level of PpUGT1 was root>leaf>flower>stem;the expression level of PpUGT7 was stem>leaf>flower>root. In addition,PpUGTs proteins were expressed in E. coli. in a soluble form.Conclusion: The genes of PpUGT1 and PpUGT7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs,and their recombinant proteins were successfully expressed in Escherichia coli. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in Paris polyphylla var. yunnanensis.

关 键 词：滇重楼 糖基转移酶 基因克隆 生物信息学分析 实时荧光定量聚合酶链式反应(Real-time PCR) 原核表达 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学]