梨树钾通道PbrAKT1的功能及其在根系钾素吸收转运中的生理作用研究

Potassium is the key nutritional element affecting fruit yield and quality of fruit trees, therefore, the potassium nutrition of fruit trees is especially concerned in agricultural production. However, the molecular mechanism of potassium uptake and transport in the root of fruit tree is still not well understood. The PbrAKT1 gene encoding potassium channel has been successfully isolated and cloned from pear root in based on previous work from the applicant, and its function trait was preliminarily studied. The functional and activated mechanism of PbrAKT1 channel is not totally similar to that for AKT1 potassium channels previously reported when tested by electrophysiological experiment. That is, the potassium uptake ability of PbrAKT1 is not regulated by proton and its activation is not required with the phosphorylation process (CBL/CIPK). Based on these fundamental results, the dual-electrode voltage clamp and patch clamp will be applied to analyze the functional characteristics and its regulatory mechanism of the PbrAKT1 channel. The response characteristics of PbrAKT1 to environmental conditions analyzed by q-PCR, upstream regulation proteins identified by yeast double impurity and BiFC experiment, the precise tissue localization of the gene and verification of the effect of its products on potassium uptake and translocation in root system under the help of model plants of Arabidopsis thaliana will also be studied in this project. Finally, it is expected to reveal the molecular mechanism of potassium uptake mediated by PbrAKT1 in the root of pear tree based on above researches, which would surely expand the understanding of the molecular mechanism of potassium nutrition in fruit plants.

钾是影响果树果实产量和品质的关键营养要素，故生产上尤为关注果树的钾素营养。然而对于果树类植物根系中钾素吸收转运的分子机理，目前还缺乏足够的理解。申请人通过前期研究已成功从梨树根系中克隆到钾通道基因PbrAKT1，并对其功能进行了初步研究。电生理实验发现该钾通道与已报道的AKT1类钾通道的作用特征和激活机制不尽相同：PbrAKT1的吸钾能力不受质子调控且其激活无需借助磷酸化过程（CBL/CIPK）。本申请拟在此基础上通过双电极电压钳和膜片钳电生理技术，深入解析PbrAKT1的功能特征及其调控机制；通过荧光定量PCR分析其对环境条件的响应特征；通过酵母双杂及BiFC实验鉴定其上游调控蛋白；借助拟南芥工具材料研究该基因的精确组织定位并验证该基因产物在根系钾吸收转运中的作用。期望通过上述研究初步揭示由PbrAKT1主导的梨树吸钾的分子机制，丰富对果树类植物钾素营养分子机制的认识。

钾是影响果树果实产量和品质的关键营养要素，故生产上尤为关注果树的钾素营养，然而对于果树类植物根系中钾素吸收转运的分子机理，目前还缺乏足够的理解。其中，Shaker家族成员是植物中研究最多的K+通道，但其在梨中的作用尚不清楚。本文以‘砀山酥梨’为材料，通过生物信息学、分子生物学和电生理技术等方法，分析了蔷薇科Shaker型通道家族成员的进化和扩张信息，以及梨PbrKAT1和PbrAKT1基因的功能及调控机制。主要研究结果如下：1）在5个蔷薇科物种中共鉴定到了36个Shaker型K+通道家族成员，依据其结构特征和系统发育分析将其分为5个亚家族。WGD事件和分散复制事件是蔷薇科Shaker型K+通道家族成员扩张的主要驱动力，且纯化选择在其进化过程中发挥主导作用。2）PbrAKT1主要在根中表达，且不受外界钾浓度的影响，PbrAKT1的吸钾能力不受质子调控且其激活无需磷酸化过程（CBL/CIPK），PbrAKT1的回补能恢复拟南芥akt1缺失突变体在低钾条件下的缺钾症状，增加植株的钾含量；荧光素酶互补试验及缺失突变体的回补试验结果显示， PbrCIPK1，PbrCIPK20和PbrCIPK27可能参与调控PbrAKT1对钾离子的吸收。3）GUS染色法及qPCR结果显示， PbrKAT1主要在叶片中表达，尤其在保卫细胞中有较高的表达；PbrKAT1能恢复缺陷型酵母菌株R5421在低钾条件下的吸钾能力；通过非洲爪蟾卵母细胞异源表达系统及双电极电压钳技术，发现PbrKAT1是一个典型的内向钾离子通道，其活性受外部钠离子的抑制，可能在梨的耐盐性调节中起重要作用。总之，本项目初步揭示了由PbrAKT1主导的梨树根系吸钾及PbrKAT1主导的梨树保卫细胞调控的分子机制，丰富了对果树类植物钾素营养分子机制的认识。