管花肉苁蓉蔗糖合酶基因的克隆鉴定、蛋白结构及表达分析

Gene cloning,functional identification,structural and expression analysis of sucrose synthase from Cistanche tubulosa

蔗糖合酶是植物糖代谢途径中的关键酶,能催化糖基供体尿苷二磷酸(uridine diphosphate,UDP)-葡萄糖的生成。本研究通过转录组数据分析从富含糖苷类化合物的传统中药管花肉苁蓉中克隆获得一个蔗糖合酶基因并命名为CtSus。该基因开放阅读框长2418 bp,编码蛋白含805个氨基酸,序列分析显示,该蛋白包含植物蔗糖合酶保守结构域,与同目植物中的蔗糖合酶具有密切的亲缘进化关系。将CtSus与已鉴定功能的糖基转移酶UGT71BD1协同进行体外全细胞转化,结果显示,CtSus的引入能显著提高UGT71BD1催化糖基化反应的转化率。利用pColdTM TF表达载体实现了CtSus在大肠杆菌中的可溶性表达,并通过体外酶促反应验证了CtSus在UDP存在的情况下催化蔗糖分解生成UDP-葡萄糖的活性。实时荧光定量PCR结果显示CtSus基因在管花肉苁蓉不同部位及干旱胁迫后肉苁蓉悬浮细胞中的表达模式与苯乙醇苷类化合物积累模式一致。进一步通过蛋白三维结构预测及分子对接探讨了该酶与底物结合的关键氨基酸及其相互作用。本研究鉴定获得了肉苁蓉中一个新的蔗糖合酶基因,为活性糖基供体的体外酶法合成提供了新的途径,亦为肉苁蓉糖苷类化合物生物合成工程菌的构建提供了新的基因元件。

Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate(UDP)-glucose,which serves as a bioactive glycosyl donor for various metabolic processes.In this study,a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa,a traditional Chinese medicine known for its rich content of diverse glycosides,based on transcriptome analysis results.The open reading frame of CtSus was 2418 bp long encoding 805 amino acids.Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein.Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order.Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1,a characterized glycosyltransferase enzyme.The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1.The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^(TM)TF expression vector.In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDPglucose in the presence of sucrose and UDP.Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C.tubulosa and the suspension cell cultures of C.tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides,respectively.Furthermore,key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results.Overall,our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C.tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.