转录因子FUSCA3在拟南芥三酰甘油积累中的功能解析

Triacylglycerol (TAG) is a main component of plant oil, which is substrate and energy source for seedling development of next generation. Understanding regulation mechanism of TAG biosynthesis is of great significance to plant developmental theory and bio-engineering of oil crops. FUSCA3 (FUS3) is a transcription factor of B3 family. As one of master regulators in Arabidopsis embryo development, FUS3 plays very important roles in accumulation of storage materials. Our latest results firstly show that FUS3 triggers TAG accumulation in vegetative cells or tissues, which provides new direct evidence to prove its role in oil accumulation. WRINKLED1 (WRI1) is an AP2 transcription factor, which is proved to be a more specific regulator of oil accumulation. Intensive studies provide insight into the regulation network of embryo-related transcription factors. FUS3 and WRI1 are proposed to be downstream regulators of LEC1 and LEC2 in the regulation network of oil accumulation. How does FUS3 regulate TAG biosynthesis? Is this regulation WRI1-dependent? They are still open questions we need to answer. Based on literature search and our transcriptome analysis of FUS3 over-expression seedlings, WRI1 is presume to be a downstream regulator of FUS3 in the process of oil accumulation. We hereby proposed a hypothesis that FUS3 activates TAG accumulation through indirect and direct effects on targets at different levels. For examples, FUS3 may activate glycolysis by WRI1, or it may direct target to promoters of seed specific genes with RY motif, like oleosins. The regulation mechanism of FUS3 on triacylglycerol will be investigated on two aspects in this proposal to test this hypothesis: Genetic interaction analysis, express pattern assay and binding assay will be employed to study the relationship between FUS3 and WRI1 during oil accumulation; ChIP-Seq, transcriptome analysis and binding assay will be conducted to find the downstream targets of FUS3 in lipid metabolism. Frame diagram of FUS3 regulation mechanism on TAG accumulation will be drawn by the combination of these results.

了解植物三酰甘油合成的调控机理对于油料作物的分子育种和基因工程改良具有极其重要的意义。转录因子FUSCA3（FUS3）是拟南芥拟南芥胚发育的主调控因子之一，在贮藏物质积累中扮演重要的角色，而在三酰甘油积累中的调控地位尚不清楚，还不了解其与WRINKLED1(WRI1)的作用关系。我们在前期研究中首次获得了FUS3过量表达能够激活三酰甘油积累的直接确凿的证据。本研究拟以此发现为出发点，从两个方面对FUS3在三酰甘油复杂的调控网络中的作用机理进行剖析：一是通过分析wri1*FUS3和WRI1*fus3的含油量变化、分析FUS3与WRI1的表达模式以及FUS3蛋白与WRI1启动子的结合，研究WRI1在FUS3调节三酰甘油积累中的作用；二是通过全基因组DNA结合位点分析、转录组分析、生物功能分析、FUS3蛋白与靶DNA体外结合、靶基因表达模式等研究，确定FUS3与三酰甘油积累有关的下游基因。

三酰甘油是种子重要的贮藏物质之一，其合成的途径已经基本清楚。然而，其调控因子种类及相互关系等调节机理还有待于研究。FUS3是控制种子发育的主调控因子，其突变体中含油量降低，但还缺乏FUS3可以诱导三酰甘油的直接证据，它与其它调控因子在三酰甘油积累过程中的关系也不清楚。本课题采用拟南芥幼苗和烟草细胞两个体系，诱导表达FUS3引发三酰甘油的明显积累，发现蔗糖在FUS3积累中扮演重要角色，在此基础上，从两个方面对FUS3调控三酰甘油的机理进行了进一步的剖析，一是对WRI1在FUS3激活三酰甘油积累过程中的作用进行研究；二是开展了FUS3与三酰甘油积累有关下游靶基因的研究。首先采用FUS3和WRI1的突变体及诱导过表达系来研究两者在三酰甘油积累中的关系。分别通过在wri1突变体中过量表达 FUS3、在fus3突变体中过量表达WRI1、FUS3与PWRI1: GUS在烟草叶片中瞬时表达等手段来确定WRI1是否处于FUS3下游。结果表明，二者在对应的突变体中表达都可以引发三酰甘油的积累，二者在三酰甘油积累中是不是简单的上下游关系，而是一种平行的关系。进一步通过研究wri1*fus3 双突变体的表型变化及在wri1和fus3突变体中对方的表达变化，发现两者表达相互影响，双突变体胚中含油量只有野生型的约5%左右，并且胚中检测到的脂肪酸主要来源于磷脂等膜脂类，表明三酰甘油积累中不同的同路主要由这两个平行调节的转录因子控制。随后通过FUS3诱导三酰甘油积累的转录组分析，我们发现了多个FUS3在调节三酰甘油积累过程中重要的下游靶位点。并在此基础上针对其中几个重要基因开展了后续研究。被FUS3强烈诱导的转录因子ABI3进一步的研究表明，ABI3也可以诱导三酰甘油的积累，结合上述结果，认为ABI3通过与其相互诱导的FUS3调节三酰甘油积累；F2-8系幼苗在FUS3表达时可以积累高达干物质5%的三酰甘油，通过分析插入位点及拷贝数，初步认为多拷贝串联插入造成FUS3高表达；另外对FUS3下游基因油体钙蛋白和DGAT1等基因的种类和功能也开展了进一步的研究。本课题在FUS3诱导三酰甘油机理方面取得的这些进展，有助于拓展对种子中三酰甘油积累调节的认识。