Author:
Su Xinglong,Meng Fei,Liu Yingying,Jiang Weimin,Wang Zhaojian,Wu Liping,Guo Xiaohu,Yao Xiaoyan,Wu Jing,Sun Zongping,Zha Liangping,Gui Shuangying,Peng Daiyin,Xing Shihai
Abstract
Platycodin D (PD) is a deglycosylated triterpene saponin with much higher pharmacological activity than glycosylated platycoside E (PE). Extensive studies in vitro showed that the transformation of platycoside E to platycodin D can be achieved using β-glucosidase extracted from several bacteria. However, whether similar enzymes in Platycodon grandiflorus could convert platycoside E to platycodin D, as well as the molecular mechanism underlying the deglycosylation process of platycodon E, remain unclear. Here, we identified a β-glucosidase in P. grandiflorus from our previous RNA-seq analysis, with a full-length cDNA of 1,488 bp encoding 495 amino acids. Bioinformatics and phylogenetic analyses showed that β-glucosidases in P. grandiflorus have high homology with other plant β-glucosidases. Subcellular localization showed that there is no subcellular preference for its encoding gene. β-glucosidase was successfully expressed as 6 × His-tagged fusion protein in Escherichia coli BL21 (DE3). Western blot analysis yielded a recombinant protein of approximately 68 kDa. In vitro enzymatic reactions determined that β-glucosidase was functional and could convert PE to PD. RT-qPCR analysis showed that the expression level of β-glucosidase was higher at night than during the day, with the highest expression level between 9:00 and 12:00 at night. Analysis of the promoter sequence showed many light-responsive cis-acting elements, suggesting that the light might regulate the gene. The results will contribute to the further study of the biosynthesis and metabolism regulation of triterpenoid saponins in P. grandiflorus.
Funder
National Natural Science Foundation of China
NSF of Anhui Province