转录因子SlIAA9影响番茄AsA积累的调控机制研究

Ascorbic acid (AsA) is an essential vitamin for people. Human can only get vitamin C from food since we have lost the capacity for AsA synthesis. Establishing tomato varieties with high AsA content via gene resources is one of the most economy and effective measures to get vitamin C. SlGMP3 is the key gene for AsA biosynthesis in tomato. In previous study, we screened the transcription factor SlIAA9 as the interacting protein of SlGMP3 promoter by yeast one-hybrid, both the mutant entire and RNAi silent material of SlIAA9 showed the significantly decreased level of AsA compared with wild type. Thus we speculated that the transcription factor SlIAA9 might affect AsA biosynthesis by regulating the expression of SlGMP3. In this project, both the transient expression system and electrophoretic mobility shift assay will be used to verify the cis-element in the SlGMP3 promoter that can be recognized and bound by SlIAA9. Then the interacting proteins of SlIAA9 will be screened by yeast two-hybrid and co-immunoprecipitation. Furthermore, the RNA-Seq of wild type, entire and SlIAA9 transgenic material will be used to reveal the regulatory network between SlIAA9 and AsA biosynthesis. Finally, this project aims to reveal the regulatory mechanism of SlIAA9 in AsA biosynthesis, and our study may provide a theoretical basis for breeding new tomato varieties with high content of AsA.

抗坏血酸（AsA）是人体所必需的维生素，人类不能合成，只能从食物中摄取，利用基因资源培育高AsA含量的番茄种质是最经济有效的手段之一。本研究前期以番茄AsA生物合成途径中的关键酶基因SlGMP3的启动子为钓饵，通过酵母单杂交筛选到转录因子SlIAA9；AsA含量在SlIAA9突变体entire和RNAi材料中明显降低，说明SlIAA9可以通过调控SlGMP3影响AsA的积累。在此基础上，本研究拟首先通过瞬时表达和电泳迁移率实验分析SlIAA9对SlGMP3启动子的识别及结合元件；然后利用酵母双杂交和免疫共沉淀等技术筛选SlIAA9的互作蛋白；最后通过RNA-Seq技术分析野生型、entire和SlIAA9转基因植株的差异表达基因，揭示SlIAA9和AsA之间的网络调控关系。综上所述，本项目的研究成果有助于揭示SlIAA9调控AsA积累的分子机制，为创制高AsA含量的番茄种质提供理论基础。

抗坏血酸（AsA）是人体所必需的维生素，人类不能合成，只能从食物中摄取，利用基因资源培育高AsA含量的番茄种质是最经济有效的手段之一。本项目首先以抗坏血酸合成途径的关键基因SlGMP3启动子为钓饵，通过酵母单杂交，从番茄cDNA文库中筛选到转录因子SlIAA9。通过检测SlIAA9的突变体entire和RNAi沉默系中AsA含量，发叶片和果实中AsA含量显著降低，同时发现SlIAA9并不能识别SlGMP3启动子，说明SlIAA9可能是间接调控SlGMP3，从而影响AsA的积累。在此研究基础上，通过酵母双杂交技术筛选到与SlIAA9互作的因子：SlARF8A和SlARF8B。进一步研究表明SlARF8A和SlARF8B可以识别并结合SlGMP3启动子，并且SlIAA9可以降低SlARF8A和SlARF8B结合SlGMP3启动子的能力。同时发现SlARF8A和SlARF8B的超量系和Crispr/Cas9敲除系中AsA含量发生了显著的变化。这些说明SlIAA9通过与SlARF8互作，进而影响SlARF8激活SlGMP3表达，最终影响AsA的积累，揭示了SlIAA9影响AsA积累的调控机制。本项目揭示SlIAA9调控AsA积累的分子机制，为创制高AsA含量的番茄新种质提供理论基础。