College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China.
State Key Laboratory of Dao-Di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Planta. 2021 Nov 30;255(1):8. doi: 10.1007/s00425-021-03797-9.
Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.
从白术中分离得到了两个鲨烯合酶基因 AlSQS1 和 AlSQS2，并通过体外酶促反应对其功能进行了表征。白术是一种传统草药，用于治疗风湿性疾病、胃病和流感。其主要活性成分包括倍半萜和三萜。鲨烯合酶（SQS；EC 2.5.1.21）催化甾醇和三萜生物合成中心异戊烯途径的第一个酶促步骤。在这项研究中，我们旨在使用克隆和体外酶特性分析来研究白术中的两个 SQS。生物信息学和系统发育分析表明，AlSQSs 与其他植物 SQSs 具有高度同源性。此外，AlSQS1 被观察到定位于细胞核和细胞质中，而 AlSQS2 则定位于细胞质和内质网中。为了获得可溶性重组酶，成功地将 AlSQS1 和 AlSQS2 作为谷胱甘肽 S-转移酶（GST）标记的融合蛋白在大肠杆菌 Transetta（DE3）中表达。使用 GST 标记亲和层析和 Western blot 分析获得约 68 kDa 的重组蛋白。体外酶反应的结果证实了 AlSQS1 和 AlSQS2 的功能，这验证了它们将两个法呢基焦磷酸转化为鲨烯的催化能力。还使用可用的 RNA 测序数据研究了两种白术化学型中 AlSQS 和选定萜类基因的表达模式。在三种组织中表达模式相对相似的 AlSQS1 和 AlSQS2 在茎中表达高于叶片和根茎。茉莉酸甲酯（MeJA）用作诱导剂分析 AlSQSs 的表达谱。qRT-PCR 分析的结果表明，AlSQS1 和 AlSQS2 的基因表达在 12 小时降至最低值，并在 24 小时达到峰值。这项研究是白术中 SQS 克隆、表征和表达的首次报道。因此，我们的研究结果提供了关于白术萜类生物合成的新见解，可能对未来的研究有用。
