杨树酚酸糖基转移酶基因PtSGT1的克隆及其酶活性鉴定

发布时间：2018-05-31 19:01

本文选题：杨树 + 酚酸类代谢　；参考：《植物生理学报》2016年06期

【摘要】：酚酸类物质在植物抵御病虫害方面发挥着重要作用,并且与木质素、类黄酮以及水杨酸等物质合成密切关联,因此,研究酚酸代谢调节具有重要意义。为了研究杨树中酚酸物质的糖基化代谢修饰,本文以毛白杨为实验材料,采用RT-PCR技术分离克隆了糖基转移酶基因PtSGT1。序列分析表明,该基因编码蛋白中含有次生代谢特征性的保守序列PSPG框。然后构建了其原核表达载体并在细菌中诱导表达,经过提取和纯化获得了PtSGT1重组蛋白。对该重组蛋白进行了广泛的酶活性筛选,对催化产物进行了色谱和质谱鉴定,发现该酶特异性地糖基化修饰酚酸类物质,而对其他类型化合物没有催化活性,证明PtSGT1是参与杨树酚酸类代谢修饰的糖基转移酶。另外,分析了PtSGT1基因在杨树9种不同组织的表达水平,发现该基因在老叶以及老茎中表达水平高,说明该糖基转移酶在体内可能与酚酸等次生代谢物的修饰、解毒、储藏等有关。本工作首次表明杨树体内存在着酚酸代谢物的糖基化修饰酶和修饰途径,为杨树的酚酸代谢调节研究以及病虫害防治奠定了基础。
[Abstract]:Phenolic acids play an important role in plant resistance to pests and diseases, and are closely related to lignin, flavonoids and salicylic acid synthesis. Therefore, it is of great significance to study the regulation of phenolic acid metabolism. In order to study the glycosylation and metabolic modification of phenolic acid in poplar, the glycosyltransferase gene PtSGT1 was isolated and cloned from Populus tomentosa by RT-PCR technique. Sequence analysis showed that the gene encoded protein contained conserved sequence PSPG frame with characteristic of secondary metabolism. Then the prokaryotic expression vector was constructed and expressed in bacteria. The recombinant PtSGT1 protein was extracted and purified. The recombinant protein was extensively screened for enzyme activity, and the catalytic product was identified by chromatography and mass spectrometry. It was found that the enzyme was specifically glycosylated to modify phenolic acids, but had no catalytic activity for other types of compounds. It is proved that PtSGT1 is a glycosyltransferase involved in the metabolic modification of poplar phenolic acids. In addition, the expression level of PtSGT1 gene in 9 different tissues of poplar was analyzed. It was found that the expression level of PtSGT1 gene was high in the old leaves and stems, indicating that the glycosyltransferase might be modified with phenolic acid and other secondary metabolites in vivo and detoxified. Relating to storage, etc. This work for the first time indicated that there were glycosylation modifiers and pathways of phenolic acid metabolites in poplar, which laid a foundation for the study of phenolic acid metabolism regulation and the control of diseases and insect pests in poplar.
【作者单位】：齐鲁师范学院生命科学学院;山东大学生命科学学院;
【基金】：国家自然科学基金(31570299) 植物细胞工程与种质创新教育部重点实验室开放课题(201103)~~
【分类号】：Q943.2;S792.11

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