Table_1_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.

香叶基焦磷酸合成酶（Farnesyl diphosphate synthase，简称FPS），作为类萜代谢途径中的关键酶，催化前体化合物香叶基焦磷酸（FPP）的合成，并在调控植物生长发育中发挥着至关重要的作用。干鳞毛蕨（Dryopteris fragrans）是一种富含类萜的药用植物。在本研究中，我们验证了该基因在体外和体内的功能，扩增了其启动子并分析了其转录活性。在本研究中，我们报道了从D. fragrans中克隆出的两个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的两个基因在配子体和成熟孢子体叶片中高表达，其表达水平在甲基茉莉酸（MeJA）和高温的刺激下增加。DfFPS的两个蛋白质均定位于细胞质中，并能在体外催化FPP的合成。我们还发现，在烟草植物中过表达DfFPS基因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，转基因植物表现出对高温和干旱的耐受性。利用融合引物和嵌套整合聚合酶链反应（FPNI-PCR）扩增了两个基因的启动子。通过烟草叶片中的β-葡萄糖苷酶（GUS）基因报告系统对启动子序列进行截断，并检测其活性，我们发现两个基因均在气孔中表达。启动子的转录活性与基因的表达模式相似，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，我们提出了对D. fragrans中FPS基因功能和转录活性的初步研究，为调控D. fragrans中的类萜代谢提供了依据。研究结果也为阐明蕨类植物中的类萜代谢途径提供了新的基础。