Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro.
10.19540/j.cnki.cjcmm.20221208.102
- Author:
Ke-Ke ZHANG
1
;
Shu-Fu SUN
2
;
Yu-Ping TAN
1
;
Zhao-Yang XU
3
;
Yin-Yin JIANG
1
;
Jian YANG
1
;
Da-Yong LI
4
;
Jin-Fu TANG
1
Author Information
1. State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700,China.
2. State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700,China School of Pharmacy,Anhui University of Chinese Medicine Hefei 230012,China.
3. College of Life Sciences,Shandong Normal University Ji'nan 250014,China.
4. Beijing Key Laboratory of Vegetable Germplasm Improvement,Beijing Vegetable Research Center,Beijing Academy of Agricultural and Forestry Sciences Beijing 100097,China.
- Publication Type:Journal Article
- Keywords:
Isatis indigotica;
chalcone isomerase;
expression analysis;
flavonoid
- MeSH:
Isatis/genetics*;
Plant Proteins/metabolism*;
Phylogeny;
Arabidopsis/genetics*;
Flavonoids;
Cloning, Molecular
- From:
China Journal of Chinese Materia Medica
2023;48(6):1510-1517
- CountryChina
- Language:Chinese
-
Abstract:
Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.
