Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. 'Splendens')
Cloning and Functional Characterization of a Gene for Capsanthin-Capsorubin Synthase from Tiger Lily (Lilium lancifolium Thunb. 'Splendens')
复制标题
DOI：
10.1093/pcp/pcs128
复制
发表时间：
2012-11-01
影响因子：
4.9
通讯作者：
Chen, Tony H. H.
中科院分区：
文献类型：
--
作者：
Jeknic, Zoran;Morre, Jeffrey T.;Chen, Tony H. H.
虎百合（Lolium lancifolium 'Splendens'）花的橙色主要是由于两种kappa-叶黄素，辣椒素和辣椒素的积累。一种被称为辣椒素-辣椒素合成酶（CCS）的酶可以催化花青素和紫黄素分别转化为辣椒素和辣椒素。采用基于番茄红素β -环化酶（lcyB）基因序列的异种非简并引物快速扩增cDNA末端（RACE）的方法，从L. lancifolium花被片中克隆了辣椒素-辣椒素合成酶（Llccs）基因。Llccs全长1785 bp，包含1425 bp的开放阅读框，编码474个氨基酸的多肽，具有预测的n端质体靶向序列。逆转录- pcr （RT-PCR）分析显示，Llccs基因的表达受时空调控，主要表达于水仙花的花蕾和花中，不表达于营养组织中。在德国鸢尾愈伤组织中稳定过表达Llccs基因，积累了包括紫黄质（capsorubin的前体）在内的多种叶黄素，导致转基因愈伤组织的颜色由正常的黄色变为红橙色。经HPLC和超高效液相色谱-串联质谱（UPLC-MS/MS）分析证实，这种新型的红橙色是由两种非天然辣椒叶黄素——辣椒素和辣椒素积累而成的。Llccs基因的克隆将促进我们对类卡帕胡萝卜素生物合成的分子和遗传机制的理解，特别是百合属植物，并将促进转基因改变许多植物物种的花和果实的颜色。
The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two kappa-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene beta-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native kappa-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of kappa-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.