Comparative analysis and natural evolution of squalene epoxidase in threeFritillariaspecies
Comparative analysis and natural evolution of squalene epoxidase in threeFritillariaspecies
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三种贝母角鲨烯环氧酶的比较分析及自然进化
DOI：
10.1007/s11103-020-01021-y
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发表时间：
2020
影响因子：
5.1
通讯作者：
Jiang Yan
中科院分区：
文献类型：
--
作者：
Lu Xu;Zhang Lin-Ning;Du Jin-Fa;Zheng Xiao-Yan;Li Hui-Jun;Li Ping;Xin Gui-Zhong;Jiang Yan
贝母是我国最常用的止咳药和食用药。建立了基于超高效液相色谱-定量-质谱（UPLC-QTOF-MS）和超高效液相色谱-定量-质谱（UPLC-QQQ-MS）的高效液相色谱-质谱（MRM）非靶向定量分析方法。ThunbergiiMiq.，F. unibractea和F.乌苏里亚种）。RNA-Seq结果表明，基因转录水平在三种贝母中具有不同的表达模式。根据转录组数据，克隆了角鲨烯环氧酶基因的全长cDNA序列。角鲨烯环氧酶基因的自然进化导致了三种贝母中的四个突变（C236 R、M489 L、G510 A和K517 R）。分子对接分析表明，236位残基位于口袋和结合中心内，其他3个残基位于蛋白表面。功能验证表明，SQE基因C236 R突变能有效提高SQE的活性，并获得较高的2，3-氧化角鲨烯产量。而SQE的突变（M489 L和G510 A）增加了蛋白表面的疏水性，也能增强SQE的活性。本研究为深入了解贝母中脂肪酸生物合成代谢产物的分化规律提供了重要的理论依据，为贝母的质量控制和代谢工程奠定了基础。
FritillariaeBulbus are the most commonly used antitussive and edible herbs in China. Based on UPLC-QTOF-MS and UPLC-QQQ-MS, the validated MRM-based non-targeted quantitative method was applied to determinate the contents of 48Fritillariaalkaloids (FAs) in threeFritillariaspecies (F. thunbergiiMiq.,F. unibracteataandF. ussuriensis). The RNA-Seq results showed that gene transcript levels have different expression patterns in threeFritillariaspecies. Based on transcriptome data, the full-length cDNA sequences of squalene epoxidase gene were cloned and characterized. Natural evolution of squalene epoxidase genes resulted in four mutations (C236R, M489L, G510A and K517R) in threeFritillariaspecies. Molecular docking analysis showed that the 236 residue is located inside the pocket and the binding center while other three residues are located on the surface of the protein. Functional verification indicated the mutations of SQE (C236R) could effectively increase the activity of SQE and obtain higher yield of 2,3-oxidosqualene in recombinant yeast. And the mutations of SQE (M489L and G510A), which increased the hydrophobicity of the protein surface, could also enhance the activity of SQE. This study provides major insights into the metabolites differentiation of FAs biosynthesis, and a firm foundation for the quality control and metabolic engineering ofFritillariaebulbus.