摘要
目的:丹参(Salvia miltiorrhiza)根中所含的丹参酮为二萜醌类化合物,而鉴定参与丹参酮合成的CYP450基因成为解析丹参酮生物合成途径分子机制的关键步骤。方法:本实验从丹参基因组和转录组数据中筛选到SmCYP81C16基因,采用RT-PCR方法克隆获得基因cDNA全长序列,利用生物信息学分析方法分析其所编码蛋白质的理化性质,预测该蛋白质二级结构、保守结构域等,建立系统发育进化树;利用实时荧光定量PCR方法检测了该基因的组织/器官表达特异性;通过遗传转化方法获得了该基因的过表达转基因毛状根株系,通过化学检测及代谢组学分析丹参酮类化合物在对照株系和过表达株系中的含量变化。利用实时荧光定量PCR方法检测了毛状根中丹参酮合成途径关键酶基因中的相对表达量。结果:SmCYP81C16基因全长1497 bp,编码498个氨基酸残基,该蛋白质相对分子质量为55.8 kDa;SmCYP81C16在丹参茎和根的周皮部高表达;通过化学检测及代谢组学分析发现,与对照株系比较,在SmCYP81C16过表达阳性株系中,丹参酮类化合物的含量升高;过表达毛状根中丹参酮合成途径关键酶基因的表达量显著上升。结论:本研究结果表明SmCYP81C16具有正向调控丹参酮生物合成的功能。本研究为利用生物技术方法提高丹参酮类化合物含量奠定基础。
Objective:Tanshinone in Salvia miltiorrhiza root is a diterpene quinone compound.Identification of the CYP450 gene involved in tanshinone synthesis has become a key step in analyzing the molecular mechanism of tanshinone biosynthesis pathway.Methods:SmCYP81C16 was screened from the genome and transcriptome data of S.miltiorrhiza and the full-length cDNA sequence was obtained and cloned by RT-PCR method.Bioinformatics analysis was used to analyze the characteristics of physicochemical properties of the coded protein,to predict the secondary structure,conserved domains,etc.of the protein,to establish phylogenetic tree;real-time quantitative PCR method was used to detect the relative expression specificity of this gene in the tissues/organs of S.miltiorrhiza.The overexpressing transgenic hairy root was obtained by genetic transformation,and the content of tanshinones was analyzed by chemical detection and metabolomics.Real-time quantitative PCR method was used to detect the comparative expression levels of key enzymes in tanshinone biosynthetic pathway in hairy roots.Results:The full-length of SmCYP81C16 was 1497 bp,encoding 498 amino acid residues.The relative molecular weight of this protein was 55.8 kDa.SmCYP81C16 was highly expressed in the stem and the periderm of S.miltiorrhiza.Through chemical detection and metabolomics analysis,it was found that compared with the control strain,in SmCYP81C16 overexpressing strains,the content of tanshinone compounds increased;the expression level of key enzyme genes of tanshinone synthesis pathway in overexpressing hairy roots increased significantly.Conclusion:SmCYP81C16 has the function of positively regulating tanshinone biosynthesis.This study lays a foundation for improving tanshinone biosynthesis content by using biotechnology.
作者
张建红
王喆
陈泓宇
严黎
吕海舟
刘琬菁
徐志超
罗红梅
ZHANG Jianhong;WANG Zhe;CHEN Hongyu;YAN Li;LYU Haizhou;LIU Wanjing;XU Zhichao;LUO Hongmei(Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Key Lab of Chinese Medicine Resources Conservation, National Administration of Traditional Chinese Medicine of the People′s Republic of China, China Academy of Medical Sciences &Peking Union Medical College, Beijing 100193, China;State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences &Peking Union Medical College, Beijing 100050, China)
出处
《世界中医药》
CAS
2020年第5期709-716,共8页
World Chinese Medicine
基金
国家自然科学基金面上项目(31570302,81973422)
中国医学科学院医学与健康科技创新工程(2016-I2M-3-016)。
