文冠果miR7760靶向MED15调控种子油脂合成积累的机制研究

Yellow horn (Xanthoceras sorbifolium) is an endemic oil-rich seed tree to China that grows well in cold, barren environments. Its seed oils are rich in oleic acid, linoleic acid, nervonic acid and other bioactive compounds with important value of medical and health care, thus are considered to be an important new healthy edible oils. However, the mechanism of miRNA mediates regulation of seed oil biosynthesis and accumulation in yellow horn is still unclear. .In plants miRNAs play important roles in the regulation of seed development and lipid metabolism by targeting regulatory protein. Mediator subunit MED15 activates the expression of lipid genes by interacting with the transcription factor WRI1 and thus affects plant seed oil biosynthesis and accumulation. On the basis of our previous work, the targeted regulating relationship between miR7760 and MED15 have been confirmed by RLM-5’RACE (RNA ligase mediated 5’rapid amplification of cDNA ends) assay. So we propose the following hypothesis that miR7760 can mediate regulation of seed oil biosynthesis and accumulation in yellow horn by targeting MED15..The main objective of this project is to test our hypothesis. Based on our already available pairs of yellow horn germplasms with close relative relationship and the significant difference in kernel oil content, the expression specificity of miR7760 in seeds at different developmental stages will be determined by analyzing the expression of GUS gene driven by the promoter of miR7760 in seeds at different developmental stages. we will adopt ultrastructural observations, Soxhlet extraction and qRT-PCR to reveal regulatory relationship between miR7760 and MED15 gene, and the regulatory role of ‘miR7760-MED15’ module in seed oil biosynthesis and accumulation according to the numbers of oil bodies, kernel oil content and expression of miR7760 and MED15. By over-expression and silencing approach, it will be identified to molecular mechanism of miR7760 targeting MED15 gene regulates oil biosynthesis and accumulation in yellow horn seeds, based on the comparative analysis of the numbers of oil bodies, kernel oil content and expression of miR7760 and MED15 between control and transformation seeds. .This project will first reveal the molecular mechanism of miR7760 targeting MED15 gene regulates seed oil biosynthesis and accumulation in woody oil-bearing plants. This will provide great significance to improve seed oil content in yellow horn.

文冠果是我国特有的木本油料树种，其种子油是重要的新型健康木本食用油，但miRNA调控其种子油脂合成积累的机制仍不清楚。围绕本项目提出的“文冠果miR7760靶向MED15调控种子油脂合成积累”假设，以近缘的高、低油文冠果种质不同发育期种子为材料，通过miR7760启动子驱动GUS基因在不同时期种子中的表达分析，明确miR7760在不同发育期种子的表达特异性；利用超微结构观测、索氏提取和qRT-PCR等手段，解析miR7760与MED15基因间的调控关系及其在油脂合成积累过程中的作用；结合过表达及沉默miR7760和MED15基因方法，比较转化与对照种子中油体数量、种仁含油率及miR7760、MED15基因表达的差异，揭示miR7760靶向MED15调控种子油脂合成积累的作用机制。这将首次揭示“miR7760-MED15”模块调控木本油料种子油脂合成积累的机理，对高油文冠果育种具有重要意义。

文冠果是我国特有的优良木本油料树种，其种子油是重要的新型健康木本食用油，但miRNA调控其种子油脂合成积累的机制仍不清楚。围绕前期研究提出的“文冠果miR7760-p5_1靶向MED15A调控种子油脂合成积累”假设，本项目以近缘、种仁含油率存在明显差异的不同文冠果种质（‘HO’和‘LO’）不同发育期种子为材料，利用索氏提取和qRT-PCR分析等技术手段，发现在种子发育过程中两个种质间miR7760-p5_1与MED15A基因的表达量呈负相关，而且MED15A基因表达量与种仁含油率呈正相关，表明miR7760-p5_1负调控靶基因MED15A的表达而影响种仁油脂的合成。烟草叶片亚细胞定位实验证实文冠果MED15A主要定位于细胞质和细胞核中。通过拟南芥遗传转化验证，发现过表达MED15A基因的拟南芥幼苗中MED15A基因、糖酵解及脂肪酸合成相关基因（PK、KAS1、FAD2和FAE1）的表达量均显著升高，暗示了文冠果MED15A基因可激活糖酵解及脂肪酸合成相关基因（即源基因）的表达，进而促进油脂的合成与积累。本项目初步阐明了miR7760-p5_1-MED15A模块在文冠果种子油脂合成与积累中的重要作用，研究结果不仅为理解木本油料种子油脂合成与积累机制提供新的理论依据，而且有助于木本油料的产油量和品质改良，同时对满足我国持续增长的优质木本粮油消费需求具有重要的理论和实践意义。