miRNA对观赏海棠叶片红色的调控机理

It is very important that the molecular regulation of anthocyanin sunthesis in Malus plant to improve fruit quality and modify the ornamental traits.Crabapple is an important ornamental and economic germplasm resource and it does not only provides us abundant plant landscape resources, but also provides favorable research material in exploiting the mechanism of color formation due to diversity in color presented in leaves, flowers and fruits. In order to know the major regulation mechanism of miRNA in ever-mauve-leaf varieties, the red-leaf variety ' Royal ' and the green-leaf cultivar 'Flame' were used as materials. We established smRNA library by high-throughput sequencing technology, analyzed the difference in expression of miRNA in leaves of different developmental stages, and forecast and find the key miRNA and its target genes related to leaf red color by Bioinformatics Methods. Furthermore, we constructed the expression vector and transformed to the callus and leaves of 'Royal' and 'Flame'. In the meantime, we determined the changes to anthocyanin content, the expression level of key enzyme gene and the main transcription factor MYB related to the anthocyanin in the transformed materials to establish molecular regulation model of 'MiRNA, target genes, constructional genes and transcriptional factors'. We look forward to providing favorable foundation in improvement color of ornamental plants.

苹果属植物花色苷合成途径的分子调控对于果实品质的提升和观赏性状的改良具有重要的作用。本研究拟以常色红叶类典型品种'王族'和常色绿叶类典型品种'火焰'为对比材料，采用高通量测序技术建立smRNA文库，分析其不同发育阶段叶片中miRNA的表达差异，初步筛选出对植物叶片花色苷合成过程中起关键作用的miRNA；然后采用生物信息学方法预测其靶标基因；在观赏海棠叶片和愈伤组织中过表达miRNA及靶标基因，分析它们的表达特性；同时结合测定和分析叶片转色过程中花色素苷含量，花色素苷代谢关键酶基因表达、主要MYB转录因子的变化，探讨叶色转变过程中相关的花色素苷代谢途径中关键miRNA与主要结构基因及转录因子的作用机制，建立'miRNA-靶标基因-转录因子-花色苷合成结构基因'的分子调控模型。旨在为苹果属植物叶色发育、品种的遗传改良提供理论基础和技术支持。

观赏海棠是果树植物中兼具观赏和经作价值的重要资源。本研究以苹果属观赏海棠常紫叶品种‘王族’和常绿叶品种‘火焰’为试材，通过small RNAs深度测序的方法，分析了其不同发育阶段叶片中miRNA的表达差异，初步筛选出了对植物叶片呈色过程中起关键作用的miRNA399d；同时发现缺P胁迫诱导miRNA399d表达，并且同时诱导其PHT1;4表达，促进CHS、ANS、MYB10等花色苷代谢途径中结构基因和转录因子的表达，同时抑制HDAC6、MET1和MYB10的表达。因此我们推测观赏海棠中mirR399d负调控花色苷的合成，是通过抑制候选靶基因PHT1;4的表达进而促进由HDAC6和MET1参与的MYB10启动子甲基化过程来实现对花色苷生物合成的负调控作用。研究结果初步揭示了观赏海棠中miRNAs对花色苷生物合成的调控机制，为苹果属植物色泽形成提供了一定的理论依据。.主要研究结果如下： （1）分别提取‘王族’和 ‘火焰’叶片small RNAs，再构建cDNA*文库，利用第二代测序技术进行深度测序。对测序结果进行一系列分析，预测miRNAs并绘制差异表达图谱，进行候选靶基因预测及其GO和KEGG分析。（2）以观赏海棠‘王族’基因组为模板，PCR克隆得到差异显著的McmiRNA159a、McmiRNA160b、McmiRNA396b、McmiRNA399d的前体序列。（3）利用瞬时转基因技术进行观赏海棠瞬时转化，得到McmiRNA159a、McmiRNA160b、McmiRNA399d的瞬时转基因株系，沉默McmiRNA159a、McmiRNA399d促进着色，沉默McmiRNA160b抑制着色。（4）分别在缺磷和磷元素正常条件下培养观赏海棠野生型和瞬时转基因植株McmiRNA399d和其经预测得到的候选靶基因McPht1;4-like株系，得到McmiRNA399d具有挽救植物缺磷症状的功能且沉默其候选靶基因McPht1;4-like也同样可以获得此效果。两种转基因株系中，经实时荧光定量PCR检测，McHDAC6表达水平均上调，花色苷积累减弱。（5）观赏海棠叶片经TSA处理之后，HDACs全面下调，呈现红色表型。构建McHDAC6瞬时沉默株系，经实时荧光定量PCR检测CHS、ANS、MYB10等基因表达水平均上调，呈现色泽加深的表型，且MYB10启动子甲基化水平下降。