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首页> 外文期刊>Journal of Experimental Botany >Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian
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Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian

机译：蓝花龙胆UDP-葡萄糖：花色素苷5-O-葡萄糖基转移酶基因的克隆与鉴定

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摘要

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.
机译：众所周知，蓝花龙胆（Gentiana triflora）会在花瓣中积聚龙胆草素，一种独特的多酰基化的delphinidin型花色苷。几乎所有参与龙胆草素生物合成的结构基因均已分离，但编码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。在这项研究中，试图分离和表征龙胆5GT，其负责花青素3-葡萄糖苷的糖基化。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，从龙胆花瓣cDNA推导的5GT基因的氨基酸序列Gt5GT7与其他植物5GT的氨基酸序列具有36.0–41.7％的同源性，系统发育分析也表明Gt5GT7属于5GT的亚科。表达分析表明，Gt5GT7转录本主要在花瓣中检测到，而在细丝中检测不到，但在叶，茎和其他花器官中未检测到。此外，花瓣中Gt5GT7转录物水平的增加与花朵的生长相吻合，这一模式与5GT酶促活性的模式相同，这是通过使用花瓣粗蛋白进行体外测定确定的。使用大肠杆菌生产的重组酶体外分析了Gt5GT7的底物特异性。 Gt5GT7可以将葡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。龙胆素的前体Delphinidin 3-glucoside是花青素3-糖苷中最好的底物。 Gt5GT7在烟草植物中的异源表达导致花青素3-芸香苷5-葡糖苷的额外积累，从而证实Gt5GT7在植物中具有有效的酶活性。

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