Gene cloning, induction, and prokaryotic expression of a Sm14-3-3 protein from Salvia miltiorrhiza.
10.19540/j.cnki.cjcmm.20220614.101
- Author:
Chen-Jing SHI
1
;
Shi-Wei WANG
2
;
Jia-Ming PENG
3
;
Hai-Yu XU
4
Author Information
1. Tianjin University of Traditional Chinese Medicine Tianjin 301617, China Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
2. School of Traditional Chinese Medicine, Guangdong Pharmaceutical University Guangzhou 510006, China.
3. School of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China.
4. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
- Publication Type:Journal Article
- Keywords:
14-3-3 protein;
Salvia miltiorrhiza;
gene cloning;
inducible expression pattern;
prokaryotic expression
- MeSH:
14-3-3 Proteins/metabolism*;
Amino Acid Sequence;
Cloning, Molecular;
Ethylenes/metabolism*;
Gene Expression Regulation, Plant;
Hormones/metabolism*;
Phylogeny;
Plant Growth Regulators/pharmacology*;
Plant Proteins/metabolism*;
Recombinant Proteins/genetics*;
Salicylic Acid/metabolism*;
Salvia miltiorrhiza/metabolism*
- From:
China Journal of Chinese Materia Medica
2022;47(18):4886-4894
- CountryChina
- Language:Chinese
-
Abstract:
14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.
