摘要
多酚氧化酶(polyphenol oxidase,PPO)是植物体内重要的抗氧化酶,它具有清除活性氧和合成酚类、木质素和植保素等功能,能增强植物抗逆和抵御病虫害能力。课题组前期研究发现丹参PPO基因能正向调控丹酚酸B合成。为深入探究其机制,该文以克隆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,验证了其无自激活、无毒性。实验室构建的丹参cDNA文库滴度为4.75×107 cfu·mL-1,符合建库要求。通过酵母双杂交试验筛选出22个可能与SmPPO互作的蛋白。通过酵母共转化验证得到只有PAL1和TAT与SmPPO互作,通过双分子荧光互补检测(BiFC)进行进一步验证,获得只有TAT与SmPPO互作,故确定TAT与SmPPO互作。分别对TAT和SmPPO按照结构域进行截短,获得SmPPO前126个氨基酸和酪氨酸氨基转移酶(TAT)在细胞膜和叶绿体上互作。通过亚细胞定位试验获得SmPPO主要定位于细胞核和细胞膜上;TAT定位于细胞膜上。实时荧光定量PCR结果表明SmPPO基因主要在根和茎中表达;TAT基因在根中表达,茎和花中表达量较低。该文为深入研究丹参SmPPO与TAT互作调控酚类物质合成的分子机制奠定了坚实基础。
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.
作者
张海星
时王珂
郭蓉
张跃进
郭宏波
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 t Yangling 712100,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2020年第11期2523-2532,共10页
China Journal of Chinese Materia Medica
基金
陕西省自然科学基金项目(2016JM8108)
中央高校基本科研业务费专项(2452016003)。
关键词
丹参
多酚氧化酶
蛋白互作
酵母双杂交
BIFC
Salvia miltiorrhiza
polyphenol oxidase
protein interaction
yeast two-hybrid
BiFC
