丛枝菌根真菌PHO1型磷转运基因在共生时期的功能研究

Phosphorus deficiency in soils has been one of the key limiting factors for the improvement of the crops yields and quality, and phosphorus is an indispensable nutrient element for plant survival and development. Arbuscular mycorrhizas (AM) is a kind of the reciprocal symbiosis formed between the soil fungi and roots of plants. The formation of AM facilitates the phosphorus uptake in plants, while the acquisition of phosphate (Pi) via the mycorrhizal uptake pathway is dependent on the phosphate transporters involved. In our previous study, we illustrated that an AM fungal PHT1-type transporter served as the Pi transceptor to sense the external Pi signal and regulate the Pi homeostasis in AM symbiosis. However, the molecular mechanism by which the phosphate is unloaded into peri-arbuscular space from arbuscules is still a puzzle. Thus, in this study, we will investigate that the PHO1-type genes from AM fungi may be involved in the phosphate unloading during AM associations established between the model AM fungi and plants and will define the molecular mechanisms by which the PHO1 genes regulate phosphate homeostasis in arbuscules through the bioinformatics, molecular and cellular biology, reverse genetics, physiological and biochemical methods. We will further analyze the binding sites between the SPX domain of PHO1 and the ligand molecule InsP6 through the protein homology modeling and the in vitro site-directed mutagenesis technology in order to study whether the SPX domain of PHO1 protein can sense the InsP6 signal and regulate the PolyPi accumulation in the yeast vacuoles. This study will uncover the mechanisms by which AM fungi control the phosphate unloading and homeostasis in arbuscules as well as provide insights into the novel biological evidence that AM fungi enhance the phosphorus absorption in plants at molecular level.

土壤缺磷已成为限制我国农作物产量与品质提高的关键因素之一，而磷是植物生长发育不可或缺的营养元素。丛枝菌根是土壤真菌与植物根系形成的一种互惠共生体，丛枝菌根的形成能促进植物对磷素的吸收，而菌根途径吸收磷主要依赖于相关的磷转运基因，前期工作阐明了丛枝菌根真菌PHT1型磷转运子作为磷转运受体感应外界磷信号，并调节丛枝菌根中磷的平衡；然而，丛枝中磷卸载进入环丛枝空间的分子机制仍然不清楚。因此，本研究将利用生物信息学、分子细胞生物学、反向遗传学及生理生化等方法，以模式丛枝菌根真菌与植物建立共生体系，研究菌根真菌PHO1型基因在菌根中卸载磷的功能及其调控丛枝中磷平衡的分子机制；利用蛋白质同源建模与体外定点诱变技术解析配体InsP6与PHO1的SPX结构域的结合位点，研究SPX结构域感应InsP6并调节酵母液泡多聚磷酸盐的积累。从分子细胞水平揭示菌根真菌调控丛枝中磷卸载与磷平衡的机制，为菌根真菌提高植物磷吸收提供新的分子生物学证据。

土壤缺磷已成为限制我国农作物产量与品质提高的关键因素之一，而磷是植物生长发育不可或缺的营养元素。丛枝菌根是土壤丛枝菌根真菌与陆生植物根系形成的一种互惠共生体，丛枝菌根的形成能促进植物对磷素的吸收。丛枝菌根真菌在共生体磷吸收与转运过程中起主导作用。前期工作阐明了丛枝菌根真菌PHT1型磷转运子作为磷转运受体感应外界磷信号；然而，丛枝中磷卸载进入环丛枝空间的分子机制仍然不清楚。因此，本研究利用生物信息学、分子细胞生物学、反向遗传学及生理生化等方法，以模式丛枝菌根真菌异形根孢囊霉或珠状巨孢囊霉与宿主植物建立共生体系，通过对异形根孢囊霉和珠状巨孢囊霉中磷信号途径的基因组和转录组分析，分离鉴定了丛枝菌根真菌中PHO1型磷转运基因RiPHO1和GmPHO1；生物信息学分析发现AM真菌的PHO1蛋白质含有SPX结构域且在真核生物中保守进化；基因表达分析发现AM真菌的RiPHO1与GmPHO1基因在丛枝中特异性表达。利用宿主和病毒诱导基因沉默技术揭示了RiPHO1或GmPHO1基因失活阻碍了丛枝菌根共生体发育，并在AM真菌组织中积累多聚磷酸盐，从而影响了菌根途径的磷吸收。研究还发现RiPHO1基因沉默激活了多聚磷酸盐合成相关基因的表达，然而抑制了多聚磷酸盐分解代谢相关基因的表达。综上所述，本研究揭示了菌根真菌PHO1型磷转运蛋白质在丛枝菌根中卸载磷酸盐的功能及其调控丛枝中磷平衡的分子机制。从分子细胞水平揭示菌根真菌调控丛枝中磷卸载与磷平衡的机制，为菌根真菌提高植物磷吸收提供了新的分子生物学证据，具有重大科学意义和理论研究价值。

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