Construction of a multigene expression system for plants and verification of its function.
10.19540/j.cnki.cjcmm.20250314.102
- Author:
Yin-Yin JIANG
1
;
Ya-Nan TANG
1
;
Yu-Ping TAN
2
;
Shu-Fu SUN
1
;
Juan GUO
1
;
Guang-Hong CUI
1
;
Jin-Fu TANG
1
Author Information
1. State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
2. State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China Experimental Research Center, China Academy of Chinese Medical Sciences Beijing 100700, China.
- Publication Type:Journal Article
- Keywords:
GFP;
betanin;
multigene expression;
transient expression;
vector construction
- MeSH:
Nicotiana/metabolism*;
Genetic Vectors/metabolism*;
Gene Expression Regulation, Plant;
Plant Proteins/metabolism*;
Plants, Genetically Modified/metabolism*;
Genetic Engineering/methods*;
Green Fluorescent Proteins/metabolism*;
Gene Expression
- From:
China Journal of Chinese Materia Medica
2025;50(12):3291-3296
- CountryChina
- Language:Chinese
-
Abstract:
Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
