Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana.
- Author:
Zhuping JIN
1
;
Cheng LI
1
;
Lei WANG
1
;
Yanxi PEI
1
Author Information
- Publication Type:Journal Article
- Keywords: Arabidopsis thaliana; epidermal pattern factor; hydrogen sulfide; protein purification; stomatal development; vector construction
- MeSH: Arabidopsis; genetics; metabolism; Arabidopsis Proteins; genetics; isolation & purification; metabolism; Escherichia coli; genetics; Genetic Vectors; genetics; Hydrogen Sulfide; metabolism; Plasmids; genetics; Seedlings; metabolism
- From: Chinese Journal of Biotechnology 2020;36(4):792-800
- CountryChina
- Language:Chinese
- Abstract: Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.
