Prokaryotic expression, subcellular localization and enzymatic activity analysis of DXS gene from Platycodon grandiflorum
10.16438/j.0513-4870.2022-0982
- VernacularTitle:桔梗DXS基因的原核表达、亚细胞定位和酶活性分析
- Author:
Nan DONG
1
;
Han-wen YU
1
;
Meng-li LIU
1
;
Jing LI
1
;
Bo-wen CHEN
1
;
Xiang-wei CHANG
1
;
Ju-tao WANG
1
;
Liang-ping ZHA
1
;
Shuang-ying GUI
1
,
2
,
3
,
4
Author Information
1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
2. Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei 230012, China
3. Engineering Technology Research Center of Modernized Pharmaceutics, Anhui Education Department (AUCM), Hefei 230012, China
4. Anhui Province Key Laboratory of Pharmaceutical Technology and Application (AUCM), Hefei 230012, China
- Publication Type:Research Article
- Keywords:
italic>Platycodon grandiflorum;
1-deoxy-D-xylulose-5-phosphate synthase;
gene cloning;
prokaryotic expression;
subcellular localization;
enzyme activity
- From:
Acta Pharmaceutica Sinica
2023;57(4):1059-1068
- CountryChina
- Language:Chinese
-
Abstract:
1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.
