Cloning and Expression Patterns of Coumarate-3-hydroxylase Gene from Angelica sinensis and Its Correlation with Content of Ferulic Acid
10.13422/j.cnki.syfjx.20221313
- VernacularTitle: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
- Author:
Manqing WANG
1
;
Xiang CHENG
1
;
Guang WANG
1
;
Yue MING
1
;
Jingyi ZENG
1
;
Xiaona BAI
1
;
Shaopeng ZHANG
1
;
Ran XU
1
Author Information
1. School of Life Science And Technology， Wuhan Polytechnic University， Wuhan 430023， China
- Publication Type:Journal Article
- Keywords:
Angelica sinensis;
ferulic acid;
coumarate-3-hydroxylase （C3H）;
gene cloning;
bioinformatics analysis;
expression analysis;
content determination
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2023;29(4):161-166
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo 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. MethodReal-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）. ResultThe open reading frame （ORF） of ASC3H （GenBank accession number： MN2550298） was 1 530 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. ConclusionASC3H 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．
