Jiang Yin-Yin, Tan Yu-Ping, Sun Shu-Fu, Tang Jin-Fu
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2022 Dec;47(24):6587-6595. doi: 10.19540/j.cnki.cjcmm.20220916.101.
Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-β-D-glucoside and lariciresinol-4'-O-β-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.
基于菘蓝的转录组数据，共鉴定出110个假定的糖基转移酶。通过原核表达和体外酶活性测定，筛选出一个新的木脂素糖基转移酶基因，命名为IiUGT349，它催化落叶松脂醇生成落叶松脂醇-4-O-β-D-葡萄糖苷和落叶松脂醇-4'-O-β-D-葡萄糖苷。生物信息学分析表明，IiUGT349包含一个1401 bp的开放阅读框（ORF），编码一个467个氨基酸的蛋白质。蛋白质分析表明，IiUGT349的预测分子量为52.77 kDa，等电点为5.96。系统发育分析表明，属于UGT90家族的IiUGT349与已报道的木脂素糖基转移酶的氨基酸序列同一性较低，可能代表一种新型的木脂素糖基转移酶。实时定量PCR（qRT-PCR）分析表明，IiUGT349在根、茎、幼叶和叶片中均有表达，在茎中的表达水平最高。进一步的生化分析表明，IiUGT349重组蛋白的最佳反应时间为12 h，最佳温度为45℃。亚细胞定位表明，IiUGT349位于植物的细胞质和细胞核中。本研究克隆了一个来自菘蓝的属于UGT90家族的新的葡萄糖基转移酶基因IiUGT349，为进一步研究其功能、阐明木脂素糖苷生物合成途径奠定了基础，对活性木脂素糖苷的合成生物学具有重要意义。
