Screening and verification of proteins of Salvia miltiorrhiza polyphenol oxidase interaction.
10.19540/j.cnki.cjcmm.20200329.103
- Author:
Hai-Xing ZHANG
1
;
Wang-Ke SHI
1
;
Rong GUO
2
;
Yue-Jin ZHANG
2
;
Hong-Bo GUO
1
Author Information
1. State Key Laboratory of Stress Biology in Arid Areas, State and Local Joint Research Center of Traditional Chinese Medicine Fingerprint and Natural Products, College of Chemistry and Pharmacy, Northwest A & F University Yangling 712100, China.
2. College of Life Sciences, Northwest A & F University Yangling 712100, China.
- Publication Type:Journal Article
- Keywords:
BiFC;
Salvia miltiorrhiza;
polyphenol oxidase;
protein interaction;
yeast two-hybrid
- MeSH:
Catechol Oxidase;
Gene Expression Regulation, Plant;
Gene Library;
Plant Proteins;
genetics;
Plant Roots;
Salvia miltiorrhiza;
genetics
- From:
China Journal of Chinese Materia Medica
2020;45(11):2523-2532
- CountryChina
- Language:Chinese
-
Abstract:
Polyphenol oxidase(PPO) is an important antioxidant enzyme in plants. It has the functions of scavenging active oxygen and synthesizing phenols, lignin, and plant protection factors, and can enhance the plant's resistance to stress and resistance to pests and diseases. Our previous research found that Salvia miltiorrhiza PPO gene can positively regulate salvianolic acid B synthesis. In order to further explore the mechanism, a pGBKT7-PPO bait vector was constructed using the cloned S. miltiorrhiza polyphenol oxidase gene(SmPPO, GenBank accession number: KF712274.1), and verified that it had no self-activation and no toxicity. The titer of S. miltiorrhiza cDNA library constructed by our laboratory was 4.75 × 107 cfu·mL~(-1), which met the requirements for library construction. Through yeast two-hybrid test, 22 proteins that could interact with SmPPO were screened. Only yeast PAL1 and TAT interacted with SmPPO through yeast co-transformation verification. Further verification was performed by bimolecular fluorescence complementary detection(BiFC). Only TAT and SmPPO interacted, so it meant that TAT and SmPPO interacted. TAT and SmPPO were truncated according to the domain, respectively. The first 126 amino acids of SmPPO and tyrosine amino transferase(TAT) were obtained to interact on the cell membrane and chloroplast. SmPPO was obtained by subcellular localization test, which was mainly loca-lized on the nucleus and cell membrane; TAT was localized on the cell membrane. Real-time quantitative PCR results showed that the SmPPO gene was mainly expressed in roots and stems; the TAT gene was expressed in roots, and the expression level in stems and flowers was low. This article lays a solid foundation for the in-depth study of the molecular mechanism of the interaction of S. miltiorrhiza SmPPO and TAT to regulate the synthesis of phenolic substances.
