《植物生理学报》 2016, 52(6): 957-966

杨树酚酸糖基转移酶基因PtSGT1的克隆及其酶活性鉴定

于惠敏^1,*, 张海龙¹, 张帜¹, 王会勇², 侯丙凯²

¹齐鲁师范学院生命科学学院, 济南250013; ²山东大学生命科学学院, 济南250100

通信作者：于惠敏；E-mail: hmyu999@163.com

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。

关键词：杨树; 酚酸类代谢; 糖基转移酶; 基因克隆

收稿：2016-04-20   修定：2016-05-13

资助：国家自然科学基金(31570299)和植物细胞工程与种质创新教育部重点实验室开放课题(201103)。 致谢 李燕洁博士和李琴硕士协助进行基因序列分析和基因表达分析, 山东大学生命科学学院公用仪器平台提供了色谱与质谱条件以及技术指导。

Cloning and enzymatic activity identification of phenolic acid glycosyltransferase gene PtSGT1 from poplar (Populus tomentosa)

YU Hui-Min^1,*, ZHANG Hai-Long¹, ZHANG Zhi¹, WANG Hui-Yong², HOU Bing-Kai²

¹School of Life Science, QiLu Normal University, Jinan 250013, China; ²School of Life Science, Shandong University, Jinan 250100, China

Corresponding author: YU Hui-Min; E-mail: hmyu999@163.com

Abstract:

Phenolic acids are thought to play an important role in resistance to diseases and pests, and also be closely related to the biosynthesis of lignins, flavonoids and salicylic acid, thus it would be valuable to study the phenolic acid metabolism regulation. In order to investigate the phenolic acid glycosylation of poplar (Populus tomentosa), a putative glycosyltransferase gene PtSGT1 was cloned from leaf cDNA of P. tomentosa by RTPCR technique. Sequence analysis indicated that the deduced protein encoded by this gene contains a consensus PSPG motif, suggesting that PtSGT1 is possibly a glycosyltransferase responsible for plant secondary metabolites. PtSGT1 gene was subcloned into prokaryotic expression plasmid and was successfully induced to express its protein products in E. coli. The recombinant protein was collected and purified. Using the recombinant protein, a widespread screening of enzymatic activity was performed toward different compounds. High Performance Liquid Chromatography (HPLC) and HPLC-Mass Spectrometry (LC-MS) analysis of reaction products indicated that PtSGT1 can only catalyze the glycosylation of phenolic acids, but not other compounds, thus demonstrating that PtSGT1 is a phenolic acid-specific glycosyltransferase of poplar. The results presented here suggest that poplar possesses the pathway of phenolic acid glycosylation in its cells. Moreover, the PtSGT1 gene has the potential to be used for metabolism research of phenolic acids and pest control in poplar.

Key words: poplar (Populus tomentosa); phenolic acid metabolism; glycosyltransferase; gene cloning