不同类型菌根中板栗PHt1家族菌根磷转运蛋白基因的功能及互作机制研究

Mycorrhizal formation and establishment is the main way of P uptake in mycorrhizal plants responsed to low phosphorus stress, which strikingly induces the expression of mycorrhizal phosphate transporter genes of PHt1 family, to transport and utilize soluble P from soil. Chestnut is a special experimental material of mycorrhizal studies in fruit trees based on that it can form both ectomycorrhizae (EM) and vesicular arbuscular (VA) mycorrhiza. So based on special mycorrhizal types of chestnut, combined with our experimental basis, and aiming at the remarkable characteristics of promoting P uptake by mycorrhiza, in this study herein, firstly we will further supplement mycorrhizal phosphate transporter genes of the PHt1 family from chestnut, and deduce evolution routes of EM and VA mycorrhiza. Secondly, RNA interference and overexpression of mycorrhizal phosphate transporter genes will be performed to study the effect on P uptake and mycorrhizal development after gene silence and overexpression, and to investigate the function of different mycorrhizal phosphate transporter genes. Thirdly, the expression of promotor::GUS/GFP will be conducted to analyze gene localization. Lastly, combined with the expression characteristics of non-mycorrhizal phosphate transporter genes in PHt1 family, we will clarify the relationship of coordinated expression among P uptake ways of direct pathway, EM mycorrhizal pathway and VA mycorrhizal pathway. The studies here has great importantance for revealing the molecular mechanism of mycorrhizal phosphate transporter genes in chestnut, and clarifying biological significance that chestnut has two kinds of mycorrhizal types.

菌根的形成和建立是菌根植物响应低磷胁迫增强磷吸收的主要途径，通过高效激活菌根中植物PHt1家族菌根磷转运蛋白基因，实现植物对土壤中可溶性磷的吸收和利用。板栗是既可形成EM外生菌根，又可形成VA内生菌根的特殊果树试材。基于板栗菌根的特殊性，结合本实验前期基础，针对菌根促磷吸收的显著特点，进一步丰富和补充PHt1家族菌根磷转运蛋白基因的不同类型，推演EM和VA菌根演化的不同路线；利用RNA干扰及超表达试验，研究菌根磷转运蛋白基因沉默和超表达后对磷吸收及菌根发育的影响，探究不同菌根磷转运蛋白基因的作用机制；构建启动子GUS/GFP载体，对基因进行细胞和亚细胞学定位；结合非菌根PHt1家族磷转运蛋白基因的时空表达特征，阐明直接、EM和VA菌根磷吸收途径中其协同表达的互作关系。这一研究对于解析板栗菌根磷转运蛋白基因作用的分子机制，以及揭示板栗具有两种菌根类型的生物学意义均具有重要作用。

菌根的形成和建立是菌根植物响应低磷胁迫增强磷吸收的主要途径，通过高效激活菌根中植物PHt1家族菌根磷转运蛋白基因，实现植物对土壤中可溶性磷的吸收和利用。板栗是我国原生干果树种，立地条件差，但由于可以形成典型的外生菌根，因此提高了板栗的抗旱和抗瘠薄能力，尤其在促磷吸收方面作用显著。本研究围绕板栗菌根磷转运蛋白基因的挖掘和功能开展研究工作，具体结果如下：. 本研究首先建立了高效的板栗菌根真菌共生体系，以此为基础，通过半薄切片显微结构分析重新界定了板栗外生菌根的不同发育阶段。其次，获得了板栗全基因组序列、板栗外生菌根转录组以及板栗菌根表皮细胞转录组数据，结合基因组和转录组以及荧光定量PCR结果，进一步丰富和补充了PHt1家族磷转运蛋白基因，分析了不同磷转运蛋白基因的作用机制；发掘了板栗菌根诱导磷转运蛋白基因CmPT5和CmPT4，CmPT5在板栗外生菌根中高效表达，负责外生菌根途径磷的吸收，CmPT4发生假基因化。. 接下来对CmPT5基因进行了启动子表达分析、亚细胞定位和基因功能研究。启动子分析发现CmPT5基因含有低磷响应P1BS和菌根诱导MYCS元件，启动子异源表达实验表明，CmPT5基因启动子在丛枝菌根植物紫云英菌根中不表达，说明其在进化过程中丢失了受丛枝菌根调控的顺式作用元件，获得了新的受外生菌根调控的顺式作用元件和转录调控因子。亚细胞定位分析发现CmPT5精准地定位在了菌根的丛枝膜上，表明板栗CmPT5可能参与了丛枝菌根中磷的转运。同时，基因功能验证也证明了CmPT5可以部分恢复苜蓿pt4突变体形成丛枝菌根的能力，这进一步证明了CmPT5为菌根磷转运蛋白基因。. CmPT5基因的挖掘和功能的解析，从分子水平上阐明了板栗外生菌根促磷吸收的机理，为后续解析CmPT5基因受外生菌根调控的机制奠定基础。该基因也成为一个可靠的标记基因，为板栗产业菌根化育苗提供关键的指纹特征。