Zhao Huan, Guo Juan, Tang Qi, Guo Lan-Ping, Huang Lu-Qi, Ma Xiao-Jun
Flow Station of Post-Doctoral Scientific Research, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Breeding Base of State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2018 Aug;43(16):3255-3262. doi: 10.19540/j.cnki.cjcmm.20180528.006.
Siraitia grosvenorii, vine plant of Cucurbitaceae family, has been used as natural sweetener and folk medicine. The major components and sweet substances are both known as mogrosides which are cucurbitane-type tetra-triterpenoids. Squalene epoxidase (SQE) has been generally recognized as the common rate-limiting enzyme in triterpenes and phytosterols, catalyzing into their common precursor 2,3-oxidosqualene (OS); however, in the biosynthesis of mogrosides, the precursor was 2,3,22,23-dioxidosqualene (DOS) instead of OS. To explore the specific SQE in S. grosvenorii, we cloned two full-length SQEs (SgSQE1, SgSQE2), performed bioinformatic analysis, analyzed the expression patterns in different periods of fruits by Real-time PCR, and induced the prokaryotic expressions. Finally, the interactive sites between SQE and substrate were predicted by docking, which would provide evidence for SQE gene function study of mogrosides and also lay foundation for triterpene biosynthesis in other plants. SgSQE1 and SgSQE2 both encoded predicted proteins of 524 amino acids, and shared 84% identity to each other at residues level, but had high specificity at N-terminal region. They both accumulated in fruits, but with different patterns, SgSQE1 increased rapidly and reached the highest level at 15 d, which had identical co-expression pattern with cucurbitadienol synthase (CS). SgSQE2 had a relatively constant level. The docking results showed that predicted proteins of SgSQE1 and SgSQE2 can interact with OS, with different contact sites (R348 for SgSQE1, H349 for SgSQE2). The recombinant proteins had no activities by prokaryotic expression, which were caused by transmembrane regions. However, all the results strongly suggested that SgSQEs were both involved in secondary metabolites biosynthesis in S. grosvenorii. SgSQE1 might be involved in mogrosides biosynthesis and SgSQE2 might participate in other cucurbitane-type triterpenes or phytosterols biosynthesis.
罗汉果是葫芦科藤本植物，一直被用作天然甜味剂和民间药物。其主要成分和甜味物质均为罗汉果甜苷，属于葫芦烷型四环三萜类化合物。角鲨烯环氧酶（SQE）通常被认为是三萜类化合物和植物甾醇生物合成中的共同限速酶，催化生成它们的共同前体2,3-氧化角鲨烯（OS）；然而，在罗汉果甜苷的生物合成中，前体是2,3,22,23-二氧化角鲨烯（DOS）而非OS。为了探究罗汉果中的特异性SQE，我们克隆了两个全长SQE（SgSQE1、SgSQE2），进行了生物信息学分析，通过实时定量PCR分析了其在果实不同时期的表达模式，并进行了原核表达诱导。最后，通过对接预测了SQE与底物之间的相互作用位点，这将为罗汉果甜苷SQE基因功能研究提供证据，也为其他植物三萜生物合成奠定基础。SgSQE1和SgSQE2均编码预测的含524个氨基酸的蛋白质，在残基水平上彼此具有84%的同一性，但在N端区域具有高度特异性。它们均在果实中积累，但模式不同，SgSQE1迅速增加并在15天时达到最高水平，其与葫芦二烯醇合酶（CS）具有相同的共表达模式。SgSQE2水平相对恒定。对接结果表明，SgSQE1和SgSQE2的预测蛋白均可与OS相互作用，接触位点不同（SgSQE1为R348，SgSQE2为H349）。原核表达的重组蛋白无活性，这是由跨膜区域导致的。然而，所有结果都强烈表明SgSQEs均参与罗汉果次生代谢产物的生物合成。SgSQE1可能参与罗汉果甜苷的生物合成，而SgSQE2可能参与其他葫芦烷型三萜类化合物或植物甾醇的生物合成。
