Department of Herbal Research, National Institute of Horticultural and Herbal Science, RDA, Eumseong 27709, Korea.
Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver, BC V6T 1Z4, Canada.
Int J Mol Sci. 2017 Dec 6;18(12):2630. doi: 10.3390/ijms18122630.
(L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3β, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3β, and C-23 remain un-conjugated.
（L）Urban 含有两种五环三萜型皂甙，即积雪草苷和羟基积雪草苷，作为主要的次生代谢产物。为了选择参与积雪草苷生物合成的 UDP-葡萄糖基转移酶（UGTs）编码候选基因，我们对甲基茉莉酸（MeJA）诱导的叶片进行了转录组分析。在这些 unigenes 中，有 120 个 isotigs 和 13 个独特序列的单核苷酸被注释为 UGTs，包括 37 个推定全长 cDNA，其中 15 个根据 UGT 委员会命名协议命名。其中之一，UGT73AH1，在 BL21（DE3）细胞中通过异源表达进行了表征。用 IPTG 诱导后，用 UDP-葡萄糖和积雪草酸对总蛋白提取物进行了测定。UPLC-QTOF/MS 分析表明，UGT73AH1 催化积雪草酸的糖基化反应，生成其单糖苷。目前尚不清楚糖基化是否发生在三萜 C-2α、C-3β、C-23 或 C-28 位上。然而，UGT73AH1 很可能在 C-28 位上进行糖基化，因为只有 C-28 位在积雪草酸的最终途径产物中带有葡萄糖部分，而 C-2α、C-3β 和 C-23 则保持未结合状态。
