Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea
10.16438/j.0513-4870.2022-0444
- VernacularTitle:茅苍术AlCMK基因的克隆及原核表达分析
- Author:
Ji-mei LU
1
;
Rui XU
1
;
Jun-xian WU
1
;
Li-si ZOU
2
;
Chao LIU
3
;
Hua-sheng PENG
1
,
4
;
Liang-ping ZHA
1
Author Information
1. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
2. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, China
3. School of Basic Medicine, Anhui Medical University, Hefei 230032, China
4. National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Science, Beijing 100700, China
- Publication Type:Research Article
- Keywords:
italic>Atractylodes lancea;
4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase;
gene cloning;
prokaryotic expression;
expression analysis;
subcellular localization
- From:
Acta Pharmaceutica Sinica
2022;57(9):2876-2884
- CountryChina
- Language:Chinese
-
Abstract:
4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK) was one of the key enzymes in the methylerythritol-4-phosphate (MEP) pathway to generate terpenoids. In this study, based on the transcriptome data of Atractylodes lancea, the sequence of the CMK gene was cloned, named AlCMK (GenBank accession number OM283293). The results showed that AlCMK contains a 1 230 bp open reading frame (ORF) encoding 409 amino acids. The deduced protein had a theoretical molecular weight of 44 752.53 and an isoelectric point of 6.67. Transmembrane structure analysis showed that there was no transmembrane structure, and the secondary structure of AlCMK was predicted to be mainly composed of random coil. Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium, Osmanthus fragrans, Eucommia ulmoides, Lonicera japonica and Salvia miltiorrhiza. Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae. The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E. coli BL21 (DE3). The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment. Meanwhile, the enzyme activity was determined by ELISA kit. The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA, and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves. The subcellular localization showed that AlCMK was located in the chloroplast. This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.
