BACKGROUND The HongXin radish (Raphanus sativus L.), which contains the natural red pigment (red radish pigment), is grown in the Fuling district of Chongqing City. However, the molecular mechanisms underlying anthocyanin synthesis for the formation of natural red pigment in the fleshy roots of HongXin radish are not well studied. RESULTS De novo transcriptome of HX-1 radish, as well as that of the advanced inbred lines HX-2 and HX-3 were characterized using next generation sequencing (NGS) technology. In total, approximately 66.22 million paired-end reads comprising 34, 927 unigenes (N50 = 1, 621 bp) were obtained. Based on sequence similarity search with known proteins, total of 30, 127 (about 86.26%) unigenes were identified. Additionally, functional annotation and classification of these unigenes indicated that most of the unigenes were predominantly enriched in the metabolic process-related terms, especially for the biosynthetic pathways of secondary metabolites. Moreover, majority of the anthocyanin biosynthesis-related genes (ABRGs) involved in the regulation of anthocyanin biosynthesis were identified by targeted search for their annotation. Subsequently, the expression of 15 putative ABRGs involved in the anthocyanin synthesis-related pathways were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Of those, RsPAL2, RsCHS-B2, RsDFR1, RsDFR2, RsFLS, RsMT3 and RsUFGT73B2-like were identified significantly associated with anthocyanin biosynthesis. Especially for RsDFR1, RsDFR2 and RsFLS, of those, RsDFR1 and RsDFR2 were highest enriched in the HX-3 and WG-3, but RsFLS were down-regulated in HX-3 and WG-3. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be act as key regulators in anthocyanin biosynthesis pathway. CONCLUSIONS The assembled radish transcript sequences were analysed to identify the key ABRGs involved in the regulation of anthocyanin biosynthesis. Additionally, the expression patterns of candidate ABRGs involved in the anthocyanin biosynthetic pathway were validated by qRT-PCR. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be acted as key regulators in anthocyanin biosynthesis pathway. This study will enhance our understanding of the biosynthesis and metabolism of anthocyanin in radish.

中文翻译：

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从头转录组萝卜（Raphanus sativus L.）肉质根的测序：分析涉及花色苷合成途径的主要基因。

背景技术含有天然红色素（红萝卜色素）的红心萝卜（Raphanus sativus L.）生长在重庆市the陵区。然而，尚未深入研究花青素合成在红心萝卜肉质根中形成天然红色素的分子机制。结果HX-1萝卜和高级自交系HX-2和HX-3的从头转录组使用下一代测序（NGS）技术进行了表征。总共获得了约3622万对配对读段，其中包含34、927个单基因（N50 = 1，621 bp）。基于与已知蛋白质的序列相似性搜索，总共鉴定出30、127（约86.26％）个单基因。此外，这些单基因的功能注释和分类表明，大多数单基因主要富含代谢过程相关的术语，尤其是次生代谢产物的生物合成途径。而且，通过有针对性地寻找它们的注释，可以鉴定出大多数与花青素生物合成相关的基因（ABRGs）。随后，使用定量实时聚合酶链反应（qRT-PCR）验证了花色苷合成相关途径中涉及的15种假定的ABRG的表达。其中，RsPAL2，RsCHS-B2，RsDFR1，RsDFR2，RsFLS，RsMT3和RsUFGT73B2-like与花色苷的生物合成显着相关。特别是对于RsDFR1，RsDFR2和RsFLS，RsDFR1和RsDFR2在HX-3和WG-3中的富集程度最高，但RsFLS在HX-3和WG-3中的表达却下调。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。结论分析了组装的萝卜转录物序列，以鉴定参与花色苷生物合成调控的关键ABRG。另外，通过qRT-PCR验证了涉及花色苷生物合成途径的候选ABRG的表达模式。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。这项研究将增进我们对萝卜中花色苷生物合成和代谢的理解。RsDFR2和RsFLS可能是花色苷生物合成途径中的关键调节剂。结论分析了组装的萝卜转录物序列，以鉴定参与花色苷生物合成调控的关键ABRG。另外，通过qRT-PCR验证了涉及花色苷生物合成途径的候选ABRG的表达模式。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。这项研究将增进我们对萝卜中花色苷生物合成和代谢的理解。RsDFR2和RsFLS可能是花色苷生物合成途径中的关键调节剂。结论分析了组装的萝卜转录物序列，以鉴定参与花色苷生物合成调控的关键ABRG。另外，通过qRT-PCR验证了涉及花色苷生物合成途径的候选ABRG的表达模式。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。这项研究将增进我们对萝卜中花色苷生物合成和代谢的理解。通过qRT-PCR验证了花色苷生物合成途径中候选ABRGs的表达模式。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。这项研究将增进我们对萝卜中花色苷生物合成和代谢的理解。通过qRT-PCR验证了花色苷生物合成途径中候选ABRGs的表达模式。我们提出，RsDFR1，RsDFR2和RsFLS的转录本可能是花色苷生物合成途径中的关键调控因子。这项研究将增进我们对萝卜中花色苷生物合成和代谢的理解。