R2R3-MYB转录因子调控杜鹃花色苷合成的分子机制研究

Flower color is one of the most important characteristics of flower quality and novel cultivar identification in ornamental plants. Rhododendron is one of the ten traditional famous flowers in China, for it has high ornamental and economic values. The Rhododendron lapponicum ‘Fuli jinling’ is a new color variation cultivar which was selected from tissue culture seedlings of ‘Jean de Marle Montague’. The results of HPLC-Q-TOF-MS analysis showed that the two cultivars contained the same compositions but significantly different contents of anthocyanins. The expression patterns of structural and regulatory genes on anthocyanin biosynthesis were analyzed by qRT-PCR. It was found that the reduced expression of regulatory genes R2R3-MYB in mutant has the mostly close relationship with the variation of flower color. However, the function and regulation mechanism of R2R3-MYB transcription factor on anthocyanin synthesis in Rhododendron is still unclear. Based on the results of the preliminary studies, RACE techniques will be used to clone R2R3-MYB from ‘Fuli jinling’ in the present project. Moreover, the expression of R2R3-MYB in different tissues and flower development stages will be analyzed by qRT-PCR, and its subcellular localization will be investigated by the transient expression in onion epidermal cells. At the same time, the promoter of R2R3-MYB protein gene will be cloned by chromosome walking technique, and the cis-regulatory elements and tissue localization will be analyzed. Furthermore, the transcriptional activation and interaction proteins of R2R3-MYB will be investigated using yeast hybrid technique. Additionally, the plant expression vector will be constructed to identify the gene function by overexpression and gene silencing. The present research will not only give new insights into the regulation mechanism of flower color variation in Rhododendron, but also provide theoretical basis for genetic improvement for flower color breeding.

花色是观赏植物的一个重要品质特征，也是新品种鉴定的重要性状之一。本研究室选育的高山杜鹃新品种‘富丽金陵’是在‘Jean de Marle Montague’组培过程中获得的花色突变体。对野生型和突变体花瓣花色苷组成进行研究，发现2个材料仅花色苷含量存在显著差异；采用定量PCR技术对花色苷合成相关基因进行表达分析，R2R3-MYB在突变体中表达下调最显著，与突变体花色改变的关系最密切。然而，R2R3-MYB转录因子在杜鹃花色苷合成中的功能和作用机制尚不清楚。因此，本项目拟在前期研究基础上，采用RACE技术克隆杜鹃R2R3-MYB基因，开展该基因的表达特征和亚细胞定位研究；采用染色体步移技术克隆启动子，进行顺式调控元件和组织定位分析；采用酵母杂交技术探讨该基因的转录激活活性和互作蛋白，并通过转基因试验进行功能验证。研究结果有助于阐明杜鹃花色变异的分子机理，为其花色性状的遗传改良提供理论依据。

花色是观赏植物的一个重要品质特征，也是新品种鉴定的重要性状之一。杜鹃花色丰富，尚不清楚花色形成的分子机制，制约了杜鹃花色的分子育种与定向遗传改良。本研究室选育的高山杜鹃新品种‘富丽金陵’是在‘Jean de Marle Montague’组培过程中获得的花色突变体，为研究高山杜鹃花色形成机理提供了宝贵的试验材料。本研究对‘富丽金陵’及野生型的花瓣花色苷组成进行研究，发现2个材料仅花色苷含量存在显著差异，明确突变体花色变异是矢车菊色素苷含量差异所致。采用第三代高通量SMRT测序技术对高山杜鹃转录组文库进行了测序，得到75002条全长转录本序列，对转录本进行功能注释、功能分类和代谢途径分析。比较分析2个材料不同花发育时期的基因表达谱，明确PAL、F3'H、DFR、ANS、MYB、bHLH基因表达协同减弱所引起的矢车菊色素苷合成受阻是突变体花色发生变异的深层次原因。其中，R2R3-MYB基因在突变体中表达下调最显著，与突变体花色改变的关系最密切。采用RACE技术克隆获得了高山杜鹃RsMYB6基因，亚细胞定位在细胞核中，在酵母体内RsMYB6具有转录激活活性，构建过量表达载体和干扰载体转化杜鹃和烟草进行功能验证。本研究结果有助于阐明杜鹃花色变异的分子机理，为其花色性状的遗传改良提供理论依据。