‘圣心’杧果实TPS13基因表达分析及酶功能验证

Expression Analysis and Functional Verification of TPS13 Gene in'Sensation'Mango Fruit

为了探索‘圣心’杧品种中萜烯合酶基因功能,本研究以‘圣心’杧果肉为实验材料,利用同源克隆技术克隆到一个具有产生萜烯类物质功能的基因,并在原核表达体系中诱导纯化出该基因的蛋白,添加前体物质进行酶促反应后,通过气相色谱质谱联用(GC-MS)上机检测该基因的产物。结果表明,在‘圣心’杧果肉中进行扩增得到一个产生萜烯类物质的基因,命名为TPS13,该基因长度在1692 bp,编码563个氨基酸,通过在线网站预测TPS13属于亲水性,不稳定蛋白,理论等电点(pI)值为5.51,说明该蛋白为酸性蛋白。通过对其结构域预测分析:TPS13蛋白的第52~540位氨基酸区间内包含一个天冬氨酸基序的cd00684结构域,属于Ⅰ类萜烯环化酶超家族。酶活性鉴定表明,该基因无法与底物GPP、FPP和GGPP生成产物,具有催化单萜类底物(NPP)生成产物柠檬烯以及α-松油醇。本试验结果为进一步研究杧果萜类物质提供参考依据。

In order to explore the function of terpene synthase gene in'Sensation'mango variety,In this study,'Sensation'mango pulp was used as the experimental material.A gene with the function of producing terpenes was cloned by homologous cloning technology,and the protein of the gene was induced and purified in the prokaryotic expression system.After adding the precursor substance for enzymatic reaction,the product of the gene was detect ed by gas chromatography-mass spectrometry(GC-MS).The results showed that a gene producing terpenes,named TPS13,could be amplified in the flesh of'Sensation'mango.The length of the gene was 1692 bp,encoding 563 amino acids.TPS13 was predicted to be hydrophilic and unstable protein by online website.The theoretical isoelectric point(pI)value was 5.51,indicating that the protein was acidic protein.By predicting its domain analysis TPS13 protein 52~540 amino acid interval contains an aspartic acid motif cd00684 domain,belonging to class I terpene cyclase superfamily.The identification of enzyme activity showed that the gene could not produce products with substrates GPP,FPP and GGPP,and could catalyze the production of limonene andα-terpineol from monoterpene substrate(NPP).This experiment provided a reference for the study of mango terpenoids.