当归香豆酸-3-羟化酶基因ASC3H的克隆及表达模式与阿魏酸含量相关性分析  

Cloning and Expression Patterns of Coumarate-3-hydroxylase Gene from Angelica sinensis and Its Correlation with Content of Ferulic Acid

作　　者：王漫青 程湘 王广 明玥 曾静怡 白小娜 张绍鹏[1] 徐燃[1] WANG Manqing;CHENG Xiang;WANG Guang;MING Yue;ZENG Jingyi;BAI Xiaona;ZHANG Shaopeng;XU Ran(School of Life Science And Technology,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生命科学与技术学院,武汉430023

出　　处：《中国实验方剂学杂志》2023年第4期161-166,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年科学基金项目(81903782);湖北省农业科技创新中心重大科技研发项目。

摘　　要：目的:对当归中香豆酸-3-羟化酶基因(C3H)全长进行克隆,进行生物信息学与基因表达模式分析,结合当归根不同组织部位阿魏酸的含量,推测ASC3H基因功能。方法:基于前期转录组测序结果,通过实时荧光定量聚合酶链式反应(Real-time PCR)克隆全长cDNA序列,运用生物信息学方法分析该序列特征,并利用Real-time PCR和高效液相色谱法(HPLC)分别测定当归不同组织中ASC3H基因表达量及阿魏酸含量。结果:克隆获得当归ASC3H全长基因(登录号MN2550298),开放阅读框(ORF)长度为1530 bp,编码509个氨基酸,理论相对分子质量为57.86 kDa,等电点8.36,为不含信号肽的亲水性不稳定蛋白,定位于叶绿体中,有跨膜区,具有多个磷酸化位点,含有细胞色素P450的保守结构域CGYDWPKGYGPIINVW_P450(383~399 aa);多重氨基酸序列比对分析结果显示ASC3H与其他植物的C3H基因具有较高相似性,并与同科植物大阿米芹的同源性最高;Real-time PCR结果显示ASC3H基因在当归不同组织的表达量不同;HPLC结果表明阿魏酸成分在当归根皮层组织含量最高,周皮、中柱次之。结论:成功获得当归C3H基因序列,分析了其蛋白结构与特异性表达特征,推测ASC3H可能参与当归阿魏酸的合成,为进一步研究C3H基因在当归阿魏酸生物途径中的功能提供了参考依据,也为遗传改良当归品质奠定了研究基础。Objective:To clone coumarate-3-hydroxylase gene(C3H)from Angelica sinensis,and analyze the correlation between its bioinformatics,expression patterns and content of ferulic acid,and to explore the functions of ASC3H.Method:Real-time polymerase chain reaction(Real-time PCR)was used to clone the full-length cDNA of ASC3H based on the transcriptome dataset of A.sinensis,and the bioinformatics analysis of the gene sequence was carried out.Real-time PCR and high performance liquid chromatography(HPLC)were used to determine relative expression of ASC3H and content of ferulic acid in different root tissues of A.sinensis(periderm,cortex and stele).Result:The open reading frame(ORF)of ASC3H(GenBank accession number:MN2550298)was 1530 bp,encoding 509 amino acids,with a theoretical molecular weight of 57.86 kDa and an isoelectric point of 8.36.It was a hydrophilic protein that was located in the chloroplast with multiple phosphorylation sites and a transmembrane region,and contained a conserved domain CGYDWPKGYGPIINVW_P450(383-399 aa)in cytochrome P450.Multiple amino acid sequence alignment analysis showed that ASC3H had high similarity with C3H from other plants,especially Ammi majus in Umbelliferae.The Real-time PCR revealed that ASC3H had different expressions in periderm,cortex and stele tissues of A.sinensis roots.It was found from HPLC that the cortex tissues had the highest content of ferulic acid,and the stele tissues had the lowest.Conclusion:ASC3H was successfully cloned from A.sinensis,and its sequence characteristics were understood more clearly,suggesting that ASC3H might be involved in the ferulic acid biosynthesis pathway of A.sinensis.This paper provided a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of ferulic acid in A.sinensis,while laying the foundation for the genetic improvement of A.sinensis.

关 键 词：当归 阿魏酸 香豆酸-3-羟化酶 基因克隆 生物信息学分析 表达分析 含量测定 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33