Abstract Celastrol with various bioactivities is a triterpenoid compound derived from Tripterygium wilfordii which is an important medicinal plant. The limited resources restrict the industrialization of celastrol. To enhance the content of celastrol, a new squalene synthase of Tripterygium wilfordii (TwSQS2) was cloned and followed by functional identification and overexpression. Recombinant TwSQS2 was obtained through prokaryotic expression and detected by SDS-PAGE and Western blot. The gas chromatograph analysis results showed that recombinant TwSQS2 converted FPP to squalene, indicating that TwSQS2 encodes a functional squalene synthase. The overexpression hairy roots were obtained by Agrobacterium rhizogenes mediated transformation and verified by β-glucuronidase histochemistry assay and genomic PCR. The enhanced expression of TwSQS2 resulted in a 2.08 fold increase of celastrol, which was up to 2.41 mg/g dry weight. These results indicated that TwSQS2 is a key enzyme in celastrol biosynthesis and its overexpression hairy roots is a promising resource to produce celastrol for replacing the natural roots. This study provides a useful genetic engineering strategy to improve the content of celastrol in Tripterygium wilfordii.

中文翻译：

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通过过表达 TwSQS2 提高雷公藤毛根培养物中 Celastrol 的产量

摘要 Celastrol 是一种具有多种生物活性的三萜化合物，来源于重要的药用植物雷公藤。有限的资源制约了celastrol的产业化。为了提高 celastrol 的含量，克隆了雷公藤新的角鲨烯合酶（TwSQS2），并进行了功能鉴定和过表达。通过原核表达获得重组TwSQS2，并通过SDS-PAGE和Western blot检测。气相色谱分析结果显示重组TwSQS2将FPP转化为角鲨烯，表明TwSQS2编码功能性角鲨烯合酶。通过发根农杆菌介导的转化获得过表达的毛状根，并通过β-葡萄糖醛酸酶组织化学测定和基因组PCR验证。TwSQS2 的增强表达导致 celastrol 增加 2.08 倍，高达 2.41 mg/g 干重。这些结果表明 TwSQS2 是 celastrol 生物合成中的关键酶，其过表达毛状根是生产 celastrol 替代天然根的有前途的资源。本研究提供了一种有用的基因工程策略，以提高雷公藤中天青素的含量。