Table_2_Functional characterization and transcriptional activity analysis of Dryopteris fragrans farnesyl diphosphate synthase genes.docx

Farnesyl diphosphate synthase (FPS), a key enzyme of the terpene metabolic pathway, catalyzes the precursor of sesquiterpene compounds farnesyl diphosphate (FPP) synthesis, and plays an important role in regulating plant growth and development. Dryopteris fragrans is a medicinal plant rich terpenoids. In this study, the function of the gene was verified in vitro and in vivo, the promoter of the gene was amplified and its transcriptional activity was analyzed. In the present study, we report the molecular cloning and functional characterization of DfFPS1 and DfFPS2, two FPS genes from D. fragrans. We found that the two genes were evolutionarily conserved. Both DfFPS genes were highly expressed in the gametophyte and mature sporophyte leaves, and their expression levels increased in response to methyl jasmonate (MeJA) and high temperature. Both DfFPS proteins were localized in the cytoplasm and could catalyze FPP synthesis in vitro. We also found that the overexpression of DfFPS genes in tobacco plants promoted secondary metabolite accumulation but exhibited negligible effect on plant growth and development. However, the transgenic plants exhibited tolerance to high temperature and drought. The promoters of the two genes were amplified using fusion primer and nested integrated polymerase chain reaction (FPNI-PCR). The promoter sequences were truncated and their activity was examined using the β-glucuronidase (GUS) gene reporter system in tobacco leaves, and we found that both genes were expressed in the stomata. The transcriptional activity of the promoters was found to be similar to the expression pattern of the genes, and the transcriptional core regions of the two genes were mainly between −943 bp and −740 bp of proDfFPS1. Therefore, we present a preliminary study on the function and transcriptional activity of the FPS genes of D. fragrans and provide a basis for the regulation of terpene metabolism in D. fragrans. The results also provide a novel basis for the elucidation of terpene metabolic pathways in ferns.

香叶基二磷酸合酶（Farnesyl diphosphate synthase，简称FPS），作为类萜代谢途径的关键酶，催化前体化合物香叶基二磷酸（FPP）的合成，并在调控植物生长与发育过程中发挥着至关重要的作用。 Dryopteris fragrans 为富含萜类化合物的药用植物。在本研究中，通过体外和体内实验验证了该基因的功能，并对其启动子进行了扩增，分析了其转录活性。本研究报道了从 D. fragrans 中克隆得到的两个 FPS 基因 DfFPS1 和 DfFPS2 的分子克隆和功能鉴定。我们发现这两个基因在进化上具有保守性。DfFPS 基因在配子体和成熟的配子体叶中高度表达，且其表达水平在甲基茉莉酸（MeJA）和高温的刺激下升高。两种 DfFPS 蛋白定位于细胞质中，并在体外能够催化 FPP 的合成。此外，我们发现过表达 DfFPS 基因的烟草植物促进了次生代谢物的积累，但对植物的生长发育影响微乎其微。然而，转基因植物对高温和干旱表现出较强的耐受性。采用融合引物和嵌套整合聚合酶链反应（FPNI-PCR）技术扩增了两个基因的启动子序列，并通过烟草叶片中的β-葡萄糖醛酸酶（GUS）基因报告系统检测了其活性，发现两个基因均表达在气孔处。启动子的转录活性与基因的表达模式相似，且两个基因的转录核心区域主要位于 proDfFPS1 的 −943 bp 至 −740 bp 之间。因此，本研究对 D. fragrans 中 FPS 基因的功能和转录活性进行了初步探讨，并为调控 D. fragrans 中的萜类代谢提供了理论依据。研究结果也为阐明蕨类植物中的萜类代谢途径提供了新的基础。