School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.
National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100007, China.
Protein Pept Lett. 2022;29(12):1061-1071. doi: 10.2174/0929866529666220831114920.
Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids.
The objective of this study was to clone and identify the PgAACT function in P. grandiflorus.
The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins.
In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems.
Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.
桔梗是亚洲著名的传统草药和功能食品，其根中以三萜类化合物为主要生物活性成分。乙酰辅酶 A C-乙酰基转移酶 (AACT) 是甲羟戊酸途径中的起始酶，在萜类化合物的生物合成中起着重要作用。
本研究旨在克隆并鉴定桔梗中的 PgAACT 功能。
通过聚合酶链反应 (PCR) 从桔梗中分离并克隆全长序列的 PgAACT 基因。使用 pET-32a 载体构建重组质粒，并在 E. coli Transetta (DE3) 细胞中表达。在烟草表皮细胞中观察 AACT 的亚细胞定位。采用定量逆转录-PCR (qRT-PCR) 鉴定 PgAACT 基因转录水平。经 MeJA 处理后，观察 AACT 基因表达的变化，并采用 UHPLC-Q-Exactive Orbitrap MS/MS 检测桔梗皂苷的变化。
本研究从桔梗中分离并克隆了两个全长 cDNA 编码 AACT1 (PgAACT1) 和 AACT2 (PgAACT2)。推导的 PgAACT1 和 PgAACT2 蛋白分别含有 408 和 416 个氨基酸。构建了重组载体，并通过优化反应条件提高了蛋白表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和 Western blot 分析表明，PgAACT 基因成功表达，重组蛋白的分子量分别为 61 和 63 kDa。亚细胞定位表明 PgAACT 基因定位于细胞质中。来自不同生境的桔梗组织特异性分析表明，PgAACT 基因在根、茎和叶中表达。经 MeJA 处理后，PgAACT 基因表达水平和桔梗总皂苷含量显著增加，表明 PgAACT 基因在调节植物防御系统中起着重要作用。
对 PgAACT1 和 PgAACT2 的克隆、表达和功能分析将有助于了解这两个基因在萜类化合物生物合成中的作用。
