丹参细胞色素P450 SmCYP72A395基因克隆及功能研究  

Isolation and functional analysis of SmCYP72A395 in Salvia miltiorrhiza

作　　者：孙琳可 张建红 王喆 陈泓宇 张文颖 郑文芳 马艺沔 何柳 孙稚颖[1] 罗红梅 SUN Lin-ke;ZHANG Jian-hong;WANG Zhe;CHEN Hong-yu;ZHANG Wen-ying;ZHENG Wen-fang;MA Yi-mian;HE Liu;SUN Zhi-ying;LUO Hong-mei(College of Pharmacy,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Engineering Research Center of Chinese Medicine Resource,Ministry of Education,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China;State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China;Department of Pharmacy,Cangzhou Central Hospital,Cangzhou 061001,China;State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]山东中医药大学药学院,山东济南250355 [2]中国医学科学院、北京协和医学院药用植物研究所,中草药物质基础与资源利用教育部重点实验室,北京100193 [3]中国医学科学院、北京协和医学院药用植物研究所,道地药材品质保障与资源持续利用全国重点实验室,北京100193 [4]河北省沧州市中心医院药学部,河北沧州061001 [5]中国医学科学院、北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,北京100050

出　　处：《药学学报》2025年第4期1166-1174,共9页Acta Pharmaceutica Sinica

基　　金：中国医学科学院医学与健康科技创新工程(2021-I2M-1-071);国家自然科学基金(31570302)。

摘　　要：基于丹参(Salvia miltiorrhiza)基因组与转录组数据,克隆获得在丹参根周皮中高丰度表达的细胞色素P450基因,命名为SmCYP72A395。采用多种在线分析工具对其编码蛋白质的理化性质、亚细胞定位、蛋白质二级结构及保守结构域进行预测分析,发现SmCYP72A395基因编码区全长为1578 bp,编码525个氨基酸残基,蛋白质分子量为59.9 kDa,理论等电点为8.68,具有一个跨膜结构域。利用实时荧光定量PCR技术检测发现SmCYP72A395在丹参花、叶和根的周皮部位表达量较高。为进一步研究该基因的生物学功能,分别构建了SmCYP72A395过表达(SmCYP72A395-OE)和RNA干扰(SmCYP72A395-RNAi)转基因毛状根材料,并利用UPLC方法检测阳性株系中丹参酮类化合物的含量。与对照株系(转化空载体的毛状根株系)相比,发现SmCYP72A395-OE株系中,二氢丹参酮I、隐丹参酮、丹参酮I及丹参酮IIA的含量均显著低于对照株系;而在SmCYP72A395-RNAi株系中,二氢丹参酮I、隐丹参酮、丹参酮I的含量显著高于对照株系。以上结果表明SmCYP72A395在丹参酮类化合物的合成积累过程起负调控作用。本研究结果为进一步阐明丹参酮类化合物生物合成及调控途径奠定基础。Based on the genome and transcriptome data of Salvia miltiorrhiza,a cytochrome P450 gene annotated as SmCYP72A395,which was highly expressed in the periderm of S.miltiorrhiza root was cloned.The total length of the cDNA sequence of SmCYP72A395 was 1578 base pairs,encoding 525 amino acids.The predicted molecular weight of the protein was 59.9 kDa.The physicochemical properties,subcellular localization,protein structure,and conserved domains were predicted by online bioinformatic tools.The theoretical isoelectric point of SmCYP72A395 was 8.68,with a transmembrane domain in the protein.This gene was highly expressed in the flower,leaf,and root periderm tissues of S.miltiorrhiza.To further identify the biological function of SmCYP72A395,the transgenic hairy roots with SmCYP72A395 overexpressed(SmCYP72A395-OE)and RNA interference(SmCYP72A395-RNAi)were constructed,and the content of tanshinone compounds was detected by UPLC between these transgenic lines and control line(haboring the vector plasmid in the transgenic line).Compared with the control line of transgenic hairy roots,the content of dihydrotanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA in the SmCYP72A395-OE lines was significantly less than those in the control line.On the contrary,the content of dihydrotanshinone I,cryptotanshinone,and tanshinone I was higher in the SmCYP72A395-RNAi lines than those in the control line.These results demonstrated that SmCYP72A395 played a negative role in the regulation of tanshinone accumulation in S.miltiorrhiza.This study lays the foundation for further elucidating the biosynthesis and regulatory pathways of tanshinone compounds in S.miltiorrhiza.

关 键 词：丹参 细胞色素P450酶 SmCYP72A395 丹参酮类化合物 生物合成调控 

分 类 号：R931[医药卫生—生药学]