苜蓿ABCG转运蛋白STR2调控共生过程中脂肪酸转运的分子机理

Most land plants can coevolve with the mycorrhizal fungi in the soil and form a mutualistic symbiosis way called Mycorrhiza Symbiosis. The exchange of nutrition is a core subject in the Mycorrhiza Symbiosis mechanism research. Previous studies of our research group have demonstrated that fatty acids are the main carbon source transferred from plants to the fungi, and we further proved that Medicago ABCG transporter STR is directly regulated by the critical transcription factor WRI5a in lipid transfer during Mycorrhizal Symbiosis. However, the function and mechanism of STR2, the homologue gene of STR, is still unknown. How is STR2 regulated by its upstream transcription factor specifically still remains elusive. Our recent studies have identified a new STR2 mutant from the Tnt1 insertion mutant library, and found that STR2 is important for the transport of fatty acid in symbiosis using the genetic transformation of hairy roots. Bioinformatic analysis result showed that the key regulatory elements of STR2 is significantly different with STR, suggesting that there exists multiple nutrient transport regulatory mechanism during Mycorrhizal Symbiosis. Based on these data, this project will comprehensively use the genetic biology, molecular biology, biochemistry biology, and bioinformatic methods, to analyse the function and mechanism of STR2 in lipid transfer during Mycorrhizal Symbiosis. We will further screen and identify the STR2 upstream transcription factor and the critical regulatory element to uncover the molecular mechanism that how the transcription of STR2 was controlled. The study of this project will reveal the molecular mechanism of nutrient exchange in the symbiotic process and construct a model for lipid transfer-related transcriptional regulatory network.

菌根共生是由植物根系与土壤中的菌根真菌形成的共生联合体，其中的营养交换机制研究是领域核心问题之一。课题组的前期研究证明了以脂肪酸为主要碳源的营养交换基础，并揭示了苜蓿ABCG转运蛋白STR受转录因子WRI5a直接调控参与共生中脂肪酸转运的机制。参与形成STR异源二聚体的STR2虽被证明参与共生，但功能尚不明确，其上游的转录调控机理也不清楚。本研究前期通过Tnt1突变体库筛选鉴定到了STR2的突变体，经毛状根遗传转化、生物信息学等方法证明STR2参与调控共生中的脂肪酸转运，且其转录相关的关键调控元件与STR有显著差异，暗示菌根共生中存在多条营养转运调控机制。申请人拟以此为切入点，综合运用遗传、分子、生化等手段，在STR2功能研究的基础上进一步鉴定STR2的上游转录因子，揭示相关的转录调控分子机制。本项目的研究对深入解析共生过程中的营养交换分子机理并构建其转录调控网络具有重要意义。

丛枝菌根（Arbuscular mycorrhizal, AM）真菌共生由球囊菌门（Glomeromycota）真菌与植物根系互作形成，AM真菌在植物根内形成高度分枝的丛枝（Arbuscule），通过这一营养交换界面，植物借磷内运蛋白MtPT4吸收真菌提供的无机磷，又将脂肪酸由ABCG半分子转运蛋白STR转给真菌供其生长所需。AP2/ERF转录因子WRI5s靶向STR和MtPT4基因启动子中的AW-box元件激活其表达，启动共生双向营养交换的开关。合成和转运脂肪酸是耗能的过程,为最大化共生效益且防止自身资源过度输出，植物需精准调节营养交换的速率和强度，相关分子机制仍存在大量未知。前人预测STR可与同源蛋白STR2形成二聚体发挥转运功能，但STR2在共生中的功能不明。. 本研究综合运用遗传学、分子生物学等方法对苜蓿ABCG转运蛋白STR2在AM共生中的功能进行了研究，鉴定了STR2上游的关键转录因子并解析了相关调控机制。本项目的结果表明，STR2参与调控AM共生丛枝发育的脂质供应。在共生早期，AP2/ERF转录因子ERM1结合STR/STR2启动子的AW-box和AW-box-like元件直接激活基因表达，与WRI5a互作共调共生的脂质转运。随着丛枝丰度增加，ERM1和WRI5a又靶向共生负调控因子ERF12启动子的DRE-box和AW-box激活其转录。ERF12既与ERM1和WRI5a互作，通过EAR基序招募辅阻遏蛋白MtTPR3a来抑制ERM1/WRI5a对STR/STR2的转录激活，又通过AP2结构域和EAR基序进行自我转录反馈抑制。. 综上所述，本研究揭示了苜蓿通过ERM1/WRI5a-ERF12转录正-负反馈环调控STR2等的表达来动态调控AM共生脂质转运的机制，完善了共生营养交换的调控网络，加深了我们对共生机理的认知，为在生产中优化共生营养交换效率提供了重要理论依据。