Zhang Hai-Xing, Shi Wang-Ke, Guo Rong, Zhang Yue-Jin, Guo Hong-Bo
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.
College of Life Sciences, Northwest A & F University Yangling 712100, China.
Zhongguo Zhong Yao Za Zhi. 2020 Jun;45(11):2523-2532. doi: 10.19540/j.cnki.cjcmm.20200329.103.
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.
多酚氧化酶（PPO）是植物中一种重要的抗氧化酶。它具有清除活性氧以及合成酚类、木质素和植物保护因子的功能，能够增强植物的抗逆性以及对病虫害的抵抗力。我们之前的研究发现，丹参PPO基因可以正向调控丹酚酸B的合成。为了进一步探究其机制，利用克隆得到的丹参多酚氧化酶基因（SmPPO，GenBank登录号：KF712274.1）构建了pGBKT7-PPO诱饵载体，并验证其无自激活且无毒性。本实验室构建的丹参cDNA文库滴度为4.75×107 cfu·mL~(-1)，满足文库构建要求。通过酵母双杂交试验，筛选出22个可与SmPPO相互作用的蛋白。经酵母共转化验证，只有酵母PAL1和TAT与SmPPO相互作用。通过双分子荧光互补检测（BiFC）进一步验证，只有TAT与SmPPO相互作用，即TAT与SmPPO相互作用。分别根据结构域对TAT和SmPPO进行截短。获得SmPPO的前126个氨基酸与酪氨酸氨基转移酶（TAT）在细胞膜和叶绿体上相互作用。通过亚细胞定位试验得出，SmPPO主要定位于细胞核和细胞膜；TAT定位于细胞膜。实时定量PCR结果显示，SmPPO基因主要在根和茎中表达；TAT基因在根中表达，但在茎和花中的表达水平较低。本文为深入研究丹参SmPPO与TAT相互作用调控酚类物质合成的分子机制奠定了坚实基础。
