miR826在芥蓝芥子油苷合成过程中的功能及其调控的分子机制

To clarify the function of miR826 in the biosynthesis of glucosinolate in Chinese kale, special vegetable in Fujian Province, the sequences of MIR826 and AOP2 were cloned, and RLM-RACE technique was used to verify the cleavage site of target gene of miR826. The transgenic plants of overexpression and silence of miR826 and AOP2 were also constructed to elucidate the role of miR826-AOP2 module in glucosinolate biosynthetic pathway. Although a lot of research focuses on the identification and function of miRNA, little information was known about the regulation of miRNA. In this project, the upstream sequences of MIR826 were cloned and cis-acting elements were predicted. Subsequently, yeast one hybrid was used to select the transcription factors functioned with cis-acting elements and the biting sites were analyzed by EMSA and ChIP. At last, the location of miR826 and AOP2 were determined by in-situ hybridization. Take it together, the upstream and downstream network of miR826 in Chinese kale is constructed and this network provides theoretical basis for transcriptional regulation of miRNA in glucosinolate biosynthesis.

本项目以福建省特色蔬菜芥蓝为研究对象，通过克隆MIR826和AOP2的完整序列，RLM-RACE进行靶基因的裂解位点验证，在芥蓝中过表达或沉默miR826来挖掘miR826在芥蓝芥子油苷合成过程中的作用。目前，鉴定和分析miRNA功能的研究很多，但对于如何调控miRNA的研究还很少，项目利用生物信息学方法结合分子生物学实验手段，克隆miR826前体合成基因MIR826上游的启动子序列，预测miR826前体的顺式作用元件，并用酵母单杂，体外EMSA和染色质免疫共沉淀ChIP等技术分析其转录调控特征，筛选影响芥蓝miR826调控芥子油苷积累的顺式作用元件和转录因子，利用原位杂交技术分析miR826和AOP2 在芥蓝中的亚细胞定位情况和组织特异性分布，构建miR826调控芥子油苷合成的上下游网络途径，为进一步通过转录手段调控miRNA在芥子油苷合成中的作用提供依据。

项目组以福建省特色蔬菜芥蓝为材料，分析了芥蓝开花后种子形成过程中芥子油苷的积累规律，关联了芥子油苷积累和种子中胚胎发育的关系，明确了鱼雷胚时期到子叶胚时期是芥子油苷积累发生显著变化的关键时期，同时，获得了种子和种荚中芥子油苷代谢路径上的相关基因，并对miR826的靶基因AOP2所在基因家族进行了分析，在众多家族成员中确定了AOP2-1为开花后芥子油苷调控的关键分子靶标。通过亚细胞定位分析AOP2主要位于细胞质，利用芥蓝AOP2抗体经过Western分析明确在植物体中的AOP2以叶片中的含量最多。对miR826和AOP2过表达植株的分析结果表明，AOP2的过量表达可以促进芥蓝中GNA含量的增加，然而miR826的过量表达却未能增加对AOP2的抑制作用，这说明在植物体内miR826对AOP2的调控还受到很多未知因素的作用。获得了芥蓝不同组织器官的混合酵母单双杂一体cDNA文库，并以miR826和AOP2的启动子构建诱饵pAbAi载体进行酵母筛库，获得了调控miR826-AOP2的候选转录因子，经过进一步基因组比对和功能分析，发现硫初生代谢路径、芥子油苷降解路径、光信号、花中特异表达的转录因子中的功能域对该组件的调控作用。以此为基础，进一步通过设置不同的硫缺乏、光差异、高温和干旱等环境条件，分析获得候选的miR826-AOP2调控元件，构建了调控miR826-AOP2合成和功能的分子网络。