Molecular Cloning and Functional Analysis of DXS and FPS Genes from Zanthoxylum bungeanum Maxim

Zanthoxylum bungeanum Maxim. (Z. bungeanum) has attracted attention for its rich aroma. The aroma of Z. bungeanum is mainly volatile terpenes synthesized by plant terpene metabolic pathways. However, there is little information on Z. bungeanum terpene metabolic gene. In this study, the coding sequence of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and farnesyl pyrophosphate synthase (FPS) were cloned from Z. bungeanum cv. ‘Fengxiandahongpao.’ ZbDXS and ZbFPS genes from Z. bungeanum with CDS lengths of 2172 bp and 1029 bp, respectively. The bioinformatics results showed that Z. bungeanum was closely related to citrus, and it was deduced that ZbFPS were hydrophilic proteins without the transmembrane domain. Subcellular localization prediction indicated that ZbDXS was most likely to be located in chloroplasts, and ZbFPS was most likely to be in mitochondria. Meanwhile, the 3D protein structure showed that ZbDXS and ZbFPS were mainly composed of α-helices, which were folded into a single domain. In vitro enzyme activity experiments showed that purified proteins ZbDXS and ZbFPS had the functions of DXS enzyme and FPS enzyme. Transient expression of ZbDXS and ZbFPS in tobacco significantly increased tobacco’s terpene content. Moreover, ZbDXS and ZbFPS were expressed in different tissues of Z. bungeanum, and the relative expression of the two genes was the highest in fruits. Therefore, this suggests that ZbDXS and ZbFPS are positively related to terpene synthesis. This study could provide reference genes for improving Z. bungeanum breeding as well as for the Rutaceae research.