雷公藤TwCYP88A1基因全长cDNA克隆与表达分析  

Cloning and Bioinformatics Analysis of TwCYP88A1 Gene in Tripterygium wilfordii

作　　者：陈上 王秀娟[1] 张逸风[1,2] 高伟[1] CHEN Shang1, WANG Xiu-juan1. , ZHANG Yi-feng1,2, GAO Wei1.(1. Beijing Key Laboratory of Traditional Chinese Medicine （TCM） Collateral Disease Theory Research, School of TCM , Capital Medical University, Beijing 100069, China ; 2. State Key Laboratory Breeding Base of Daodi Herbs, National Resourse Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China)

机构地区：[1]首都医科大学中医药学院,中医络病研究北京市重点实验室,北京100069 [2]中国中医科学院中药资源中心道地药材国家重点实验室培育基地,北京100700

出　　处：《中国实验方剂学杂志》2018年第6期32-36,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金优秀青年基金项目(81422053);国家自然科学基金面上项目(81373906)

摘　　要：目的：研究雷公藤萜类成分的生物合成相关基因,在雷公藤转录组信息数据的基础上,以雷公藤悬浮细胞为研究材料,克隆Tw CYP88A1基因。方法：采用c DNA末端快速扩增技术（RACE）克隆雷公藤Tw CYP88A1基因,实时荧光定量聚合酶链式反应（PCR）分析其组织表达谱。结果：Tw CYP88A1 c DNA全长1 594 bp,编码491个氨基酸,等电点9.16,相对分子质量55.957 k Da,具有半胱氨酸铁血红素配体信号活性催化位点。植物组织表达分析表明Tw CYP88A1基因在雷公藤叶中的表达量最高,根中最低。结论：从雷公藤悬浮细胞中克隆得到Tw CYP88A1基因全长c DNA,对其进行生物信息学分析,揭示其组织表达谱,为深入分析雷公藤细胞色素P450酶、解析雷公藤萜类化合物的生物合成下游途径奠定基础。Objective： To study on the genes related to the biosynthesis of diterpenoid in Tripterygium wilfordii,based on the transcriptome data, TwCYP88 A1 gene was cloned from T. wilfordii suspension cells.Method： TwCYP88 A1 was obtained by rapid amplification of c DNA ends（ RACE）. Real-time quantitative polymerase chain reaction（ PCR） was used to detect the relative expression profile of TwCYP88 A1 in different tissues of T. wilfordii. Result： The full-length c DNA of TwCYP88 A1 was 1 594 bp encoding a polypeptide of 491 amino acids. The deduced isoelectric point（ p I） was 9. 16, a calculated relative molecular weight was about55. 957 k Da. It had active catalytic sites of cysteine hemoglobin ligand signal. Plant tissue expression analysis indicated that TwCYP88 A1 expressed the highest in leaves,while lowest in roots. Conclusion： The full-length c DNA of TwCYP88 A1 can be cloned from the suspension cells of T. wilfordii.

关 键 词：雷公藤 TwCYP88A1 组织表达 聚合酶链式反应 悬浮细胞 邻接法 细胞色素P450 

分 类 号：R282[医药卫生—中药学] R931[医药卫生—中医学]