Construction of prokaryotic expression and over-expression vectors of squalene epoxidase gene from Sanghuangporus baumii
10.7501/j.issn.0253-2670.2018.11.023
- Author:
Ting-Ting SUN
1
Author Information
1. Department of Food Engineering, Harbin University
- Publication Type:Journal Article
- Keywords:
PCR;
Prokaryotic expression;
Sanghuangporus baumii (Pilát) L. W. Zhou & Y. C. Dai;
Squalene epoxidase;
Vector construction
- From:
Chinese Traditional and Herbal Drugs
2018;49(11):2632-2639
- CountryChina
- Language:Chinese
-
Abstract:
Objective To obtain prokaryotic expression and over-expression vectors of squalene epoxidase (SE) gene from Sanghuangporus baumii. Methods The entire protein-coding cDNA of SE was cloned into the expression vector pET-32a (+). Then the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. SDS-PAGE was used to investigate the situation of expression after IPTG induction for 2-10 h. Additionally, primers were designed according to the gpd promoter sequence of Lentinula edodes in GenBank, and the gpd promoter fragment was obtained by PCR. Subsequently, the plant binary expression vector pCAMBIA1301 was selected as the basic vector, and then the 35 S promoter replaced with L. edodes gpd promoter through enzyme digestion and connection. Finally, the coding region of SE was cloned to the downstream of the gpd promoter to construct over-expression vectors. Results The prokaryotic expression vector pET-32a-SE was successfully obtained. SDS-PAGE results showed a significant protein band was found in the vicinity of the relative molecular weight of approximately 55 000, consistent with molecular weight of the predicted protein. Moreover, the over-expression vector pCAMBIA1301-gpd-gpd-SE was constructed successfully through different detection ways. Conclusion These results lay the foundation for the further study of SE in triterpenoid biosynthesis pathway of S. baumii.
