Equipe Physiologie, Pathologie et Génétique Végétales (PPGV), INP-PURPAN, Université de Toulouse, 75 voie du TOEC, BP 57611, 31076, Toulouse Cedex 03, France.
Laboratoire de Recherche en Sciences Végétales (LRSV), CNRS, Université Paul Sabatier (UPS), Toulouse, France.
Sci Rep. 2020 Sep 15;10(1):15137. doi: 10.1038/s41598-020-71746-9.
Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.
糖基化是一种关键的修饰方式，它有助于决定植物天然产物（包括萜类化合物和甜菊糖苷）的生物活性和生物利用度。它是由尿苷二磷酸糖基转移酶（UGTs）介导的，UGTs 通过将糖转移到小分子上来实现其活性。因此，甜菊糖苷的多样性归因于甜菊醇 C-13 和 C-19 羟基上糖基化的数量、位置和性质。尽管甜菊糖苷具有强烈的甜味特性，并且已经进行了大量研究，但甜菊糖苷的生物合成途径仍在很大程度上未知。在甜叶菊中已经鉴定出超过 60 种甜菊糖苷和 68 种推定的 UGT。本研究旨在提供方法来鉴定可能参与甜菊糖苷生物合成的 UGT。在农杆菌介导的瞬时基因表达后，可在表达重组 UGT 的植物中或从粗提物中简单直接地测试其功能。从 pGWBs 系列的二元载体组合使用来生产含有甜叶菊 UGT 的表达载体，可实现对多种底物的功能测试以及其他进一步分析的应用，包括亚细胞定位。
