Main conclusionA high level of the secondary metabolite chicoric acid is produced by intracellular Pi supply and extracellular phosphate limiting in Echinacea purpurea hairy roots.AbstractChicoric acid (CA) is a secondary metabolite which is gained from Echinacea purpurea. It has been found to be one of the most potent HIV integrase inhibitors with antioxidant and anti-inflammatory activities. However, the low-biosynthesis level of this valuable compound becomes an inevitable obstacle limiting further commercialization. Environmental stresses, such as phosphorus (Pi) deficiency, stimulate the synthesis of chemical metabolites, but significantly reduce plant growth and biomass production. To overcome the paradox of dual opposite effect of Pi limitation, we examined the hypothesis that the intracellular Pi supply and phosphate-limiting conditions enhance the total CA production in E. purpurea hairy roots. For this purpose, the coding sequence (CDS) of a purple acid phosphatase gene from Arabidopsis thaliana, AtPAP26, under CaMV-35S promoter was overexpressed in E. purpurea using Agrobacterium rhizogenes strain R15834. The transgenic hairy roots were cultured in a Pi-sufficient condition to increase the cellular phosphate metabolism. A short-term Pi starvation treatment of extracellular phosphate was applied to stimulate genes involved in CA biosynthesis pathway. The overexpression of AtPAP26 gene significantly increased the total APase activity in transgenic hairy roots compared to the non-transgenic roots under Pi-sufficient condition. Also, the transgenic hairy roots showed increase in the level of total and free phosphate, and in root fresh and dry weights compared to the controls. In addition, the phosphate limitation led to significant increase in the expression level of the CA biosynthesis genes. Considering the increase of biomass production in transgenic vs. non-transgenic hairy roots, a 16-fold increase was obtained in the final yield of CA for transgenic E. purpurea roots grown under −P condition compared to +P non-transgenic roots. Our results suggested that the expression of phosphatase genes and phosphate limitation were significantly effective in enhancing the final production yield and large-scale production of desired secondary metabolites in medicinal plant hairy roots.

中文翻译：

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酸性磷酸酶基因的异源表达和磷酸盐限制导致紫锥菊转基因毛状根中大量生产菊苣酸

主要结论紫锥菊毛状根的胞内供磷和胞外限磷作用会产生高水平的次生代谢产物菊苣酸。摘要菊苣酸(Chicoric acid,CA)是紫锥菊的次级代谢产物。已发现它是最有效的 HIV 整合酶抑制剂之一，具有抗氧化和抗炎活性。然而，这种有价值的化合物的低生物合成水平成为限制进一步商业化的不可避免的障碍。环境压力，例如磷 (Pi) 缺乏，会刺激化学代谢物的合成，但会显着降低植物生长和生物量生产。为了克服 Pi 限制的双重相反效应的​​悖论，我们检验了细胞内 Pi 供应和磷酸盐限制条件增强 E. purpurea 毛状根中总 CA 产量的假设。为此目的，使用发根农杆菌菌株 R15834，在 CaMV-35S 启动子下来自拟南芥的紫色酸性磷酸酶基因 AtPAP26 的编码序列 (CDS) 在 E. purpurea 中过表达。在磷充足的条件下培养转基因毛状根以增加细胞磷酸盐代谢。应用细胞外磷酸盐的短期 Pi 饥饿处理来刺激参与 CA 生物合成途径的基因。在 Pi 充足条件下，与非转基因根相比，AtPAP26 基因的过表达显着增加了转基因毛根中的总 APase 活性。还，与对照相比，转基因毛状根的总磷酸盐和游离磷酸盐水平以及根的鲜重和干重均有所增加。此外，磷酸盐限制导致 CA 生物合成基因的表达水平显着增加。考虑到转基因毛状根与非转基因毛状根中生物量产量的增加，与+P 非转基因根相比，在-P 条件下生长的转基因 E. purpurea 根的 CA 最终产量增加了 16 倍。我们的研究结果表明，磷酸酶基因的表达和磷酸盐限制在提高药用植物毛状根中所需次生代谢物的最终产量和大规模生产方面显着有效。此外，磷酸盐限制导致 CA 生物合成基因的表达水平显着增加。考虑到转基因毛状根与非转基因毛状根中生物量产量的增加，与+P 非转基因根相比，在-P 条件下生长的转基因 E. purpurea 根的 CA 最终产量增加了 16 倍。我们的研究结果表明，磷酸酶基因的表达和磷酸盐限制在提高药用植物毛状根中所需次生代谢物的最终产量和大规模生产方面显着有效。此外，磷酸盐限制导致 CA 生物合成基因的表达水平显着增加。考虑到转基因毛状根与非转基因毛状根中生物量产量的增加，与+P 非转基因根相比，在-P 条件下生长的转基因 E. purpurea 根的 CA 最终产量增加了 16 倍。我们的研究结果表明，磷酸酶基因的表达和磷酸盐限制在提高药用植物毛状根中所需次生代谢物的最终产量和大规模生产方面显着有效。与 +P 非转基因根相比，在 -P 条件下生长的转基因 E. purpurea 根的 CA 最终产量增加了 16 倍。我们的研究结果表明，磷酸酶基因的表达和磷酸盐限制在提高药用植物毛状根中所需次生代谢物的最终产量和大规模生产方面显着有效。与 +P 非转基因根相比，在 -P 条件下生长的转基因 E. purpurea 根的 CA 最终产量增加了 16 倍。我们的研究结果表明，磷酸酶基因的表达和磷酸盐限制在提高药用植物毛状根中所需次生代谢物的最终产量和大规模生产方面显着有效。