中国中药杂志

2017, v.42(02) 220-225

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雷公藤牻牛儿基焦磷酸合酶基因TwGPPS克隆与表达分析
Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii

屠李婵;张逸风;苏平;胡添源;童宇茹;关红雨;赵瑜君;张夏楠;袁媛;高伟;黄璐琦;
TU Li-chan;ZHANG Yi-feng;SU Ping;HU Tian-yuan;TONG Yu-ru;GUAN Hong-yu;ZHAO Yu-jun;ZHANG Xia-nan;YUAN Yuan;GAO Wei;HUANG Lu-qi;School of Traditional Chinese Medicine,Capital Medical University;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,Chinese Academy of Chinese Medical Sciences;

摘要(Abstract):

根据雷公藤悬浮细胞转录组数据设计特异引物,采用cDNA末端快速扩增技术克隆雷公藤牻牛儿基焦磷酸合酶基因TwGPPS1,TwGPPS2全长cDNA,并进行生物信息学分析和蛋白表达研究。首次克隆得到TwGPPS1核苷酸的完整开放阅读框长度为1 278 bp,编码425个氨基酸蛋白,预测蛋白等电点为6.68,相对分子质量为47.189 kDa;TwGPPS2核苷酸的完整开放阅读框长度为1 269 bp,编码422个氨基酸蛋白,预测蛋白等电点为6.71,相对分子质量为46.774 kDa;进一步构建了原核重组表达载体pET-32a-TwGPPS1,pET-32a-TwGPPS2,并成功诱导出TwGPPS1,TwGPPS2重组蛋白,为深入研究此关键酶基因功能和雷公藤萜类生物合成途径奠定基础。
Based on the transcriptome data,the study cloned full-length c DNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a1 278 bp open reading frame( ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point( p I) was 6. 68,a calculated molecular weight was about 47. 189 k Da. The full-length c DNA of the TwGPPS2 contains a 1 269 bp open reading frame(ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point(p I) was 6. 71,a calculated molecular weight was about 46. 774 k Da. The entire reading frame of TwGPPS1,2 was cloned into the p ET-32 a( +) vector and expressed in E. coli BL21( DE3) cells to obtain the TwGPPS protein,which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

关键词(KeyWords): 雷公藤;牻牛儿基焦磷酸合酶;克隆;生物信息学分析;蛋白表达
Tripterygium wilfordii;geranyl diphosphate synthase;cloning;bioinformatics analysis;protein expression

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基金项目(Foundation): 北京高等学校高水平人才交叉培养“实培计划”项目;; 国家自然科学基金优秀青年科学基金项目(81422053)

作者(Author): 屠李婵;张逸风;苏平;胡添源;童宇茹;关红雨;赵瑜君;张夏楠;袁媛;高伟;黄璐琦;
TU Li-chan;ZHANG Yi-feng;SU Ping;HU Tian-yuan;TONG Yu-ru;GUAN Hong-yu;ZHAO Yu-jun;ZHANG Xia-nan;YUAN Yuan;GAO Wei;HUANG Lu-qi;School of Traditional Chinese Medicine,Capital Medical University;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,Chinese Academy of Chinese Medical Sciences;

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