Identification and Characterization of R2R3-MYB and bHLH Transcription Factors Regulating Anthocyanin Biosynthesis in Gentian Flowers
Identification and Characterization of R2R3-MYB and bHLH Transcription Factors Regulating Anthocyanin Biosynthesis in Gentian Flowers
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DOI：
10.1093/pcp/pcn163
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发表时间：
2008-12-01
影响因子：
4.9
通讯作者：
Nishihara, Masahiro
中科院分区：
文献类型：
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作者：
Nakatsuka, Takashi;Haruta, Katia Sanae;Nishihara, Masahiro
甘草植物有鲜艳的蓝色花朵，这是由多酰化花青素龙胆草黄素的积累引起的。我们以前进行了表达分析的龙胆草生物合成基因，并假设，白色花龙胆品种Polarno白色可能是由于突变的某些调控因子负责花青素在花瓣中的生物合成。本研究采用简并PCR和cDNA末端快速扩增（RACE）技术，从紫花龙胆中分离到26个R2 R3-MYB基因片段，包括4个全长cDNA（GtMYB 2a、GtMYB 2b、GtMYB 3和GtMYB 4）和1个碱性螺旋环螺旋（bHLH）基因（GtbHLH 1）。系统进化树分析表明，GtMYB 3与矮牵牛AN 2和拟南芥PAP 1同属一个花色素苷合成分支。另一方面，基于系统发育和基因组结构分析，GtbHLH 1与矮牵牛AN 1表现出高度的同源性。GtMYB 3和GtbHLH 1转录水平的时间分布与花瓣中龙胆草素积累及其生物合成基因的时间分布相对应。酵母双杂交分析表明GtbHLH 1与GtMYB 3相互作用。此外，瞬时表达分析表明，GtMYB 3和GtbHLH 1的共表达可以增强烟草BY 2细胞中晚期花色素苷生物合成基因的启动子活性。我们还发现，在CV。Polarno白色：GtMYB 3基因因插入转座因子或未表征序列而发生突变，表明白色着色是由GtMYB 3突变引起的。这些结果表明GtMYB 3和GtbHLH 1参与了龙胆花中龙胆草素生物合成的调控。
Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin gentiodelphin. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar Polarno White might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helixloophelix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. Polarno White the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.