Nakatsuka Takashi, Sato Kei, Takahashi Hideyuki, Yamamura Saburo, Nishihara Masahiro
Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate 024-0003, Japan.
J Exp Bot. 2008;59(6):1241-52. doi: 10.1093/jxb/ern031. Epub 2008 Mar 28.
Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0-41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.
已知蓝花龙胆（Gentiana triflora）在花瓣中积累龙胆花色素苷，这是一种独特的多酰化飞燕草素型花青素。几乎所有参与龙胆花色素苷生物合成的结构基因都已被分离出来，但一个编码UDP-葡萄糖：花青素5-O-葡萄糖基转移酶（5GT）的重要基因仍有待鉴定。在本研究中，我们试图分离和鉴定负责花青素3-葡萄糖苷糖基化的龙胆5GT。基于PCR的克隆策略从龙胆花中鉴定出7个5GT候选基因。其中，来自龙胆花瓣cDNA的5GT基因（命名为Gt5GT7）推导的氨基酸序列与其他植物物种的5GT具有36.0-41.7%的同一性，系统发育分析也表明Gt5GT7属于5GT亚家族。表达分析表明，Gt5GT7转录本主要在花瓣中检测到，在花丝中较弱，但在叶、茎和其他花器官中未检测到。此外，花瓣中Gt5GT7转录本水平的增加与花的发育一致，这一模式与使用花瓣粗蛋白进行体外测定所确定的5GT酶活性模式相同。使用大肠杆菌产生的重组酶在体外分析了Gt5GT7的底物特异性。Gt5GT7可以将葡萄糖基部分转移到花青素3-糖苷上，但不能转移到其他类黄酮化合物上。在测试的几种花青素3-糖苷中，龙胆花色素苷的前体飞燕草素3-葡萄糖苷是最佳底物。Gt5GT7在烟草植物中的异源表达导致矢车菊素3-芸香糖苷-5-葡萄糖苷的额外积累，证实Gt5GT7在植物中具有有效的酶活性。
