Cloning and expression analysis of 2-oxoglutarate-dependent dioxygenase from Schisandra chinensis
10.16438/j.0513-4870.2020-1068
- VernacularTitle:五味子2-酮戊二酸依赖性双加氧酶基因的克隆及表达分析
- Author:
Hai-yan LI
1
;
Jiu-shi LIU
2
;
Ting WANG
1
;
Yu-yang LIU
1
;
Xi-ang WANG
1
;
Hong-bo LI
1
Author Information
1. College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
2. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
- Publication Type:Research Article
- Keywords:
italic>Schisandra chinensis;
Sc2-ODD;
gene cloning;
qRT-PCR;
prokaryotic expression;
protein purification
- From:
Acta Pharmaceutica Sinica
2020;55(9):2226-2233
- CountryChina
- Language:Chinese
-
Abstract:
The 2-oxoglutarate-dependent dioxygenase (2-ODD) gene is regarded as the key enzyme gene involved with aryl naphthalene lignan-podophyllotoxin synthesis. To study the expression pattern and function of the Sc2-ODD gene, a full-length cDNA of the gene was cloned. Bioinformatic analysis, the expression pattern, and prokaryotic expression and purification were implemented. The open reading frame of Sc2-ODD gene was 1 077 bp and encoded 358 amino acids with a molecular weight of 40.16 kD. The Sc2-ODD protein contained the conserved 2OG-FeII-oxy sequence of the 2-ODD protein. The results of phylogenetic analysis revealed that Sc2-ODD is most closely related to Corchorus olitorius 2-ODD. qRT-PCR results showed that Sc2-ODD expression displayed obvious up-regulation at the fruit-swelling stage, then down-regulation in the fruit-coloring period. The Sc2-ODD gene was cloned into the bacterial expression vector pGS21T, the recombinant Sc2-ODD protein was expressed in Escherichia coli Rosetta (DE3) cells and the fusion protein was obtained and purified by GST fusion protein purification technology. This study will lay a foundation for further research on the function and expressional regulation of the Sc2-ODD gene in the aryl naphthalene lignans biosynthesis pathway, and also provides a scientific basis for improving the lignan content and the medicinal quality of Schisandra chinensis using plant genetic engineering.
