柱花草紫色酸性磷酸酶SgPAP23参与植酸磷活化利用的分子机理

Stylo (Stylosanthes spp.) is a dominant leguminous forage in the tropical and subtropical areas. Under low phosphorus (P) condition, stylo has superior capability to utilize phytate, but the molecular mechanisms underlying stylo high efficient utilization of phytate remain largely unknown. It has been demonstrated that plant purple acid phosphatase (PAP) play a vital role in the utilization and mobilization of phytate. In our previous study, a novel PAP, SgPAP23, was characterized inducing by P deficiency in stylo. Overexpression of SgPAP23 resulted in significantly increased phytate utilization in the transgenic Arabidopsis. In the present study, we plan to investigate the expression patterns of SgPAP23 in response to P deficiency in stylo at both transcription and protein levels through Real-time PCR and Western-blot analysis. Subsequently, to evaluate functions of SgPAP23 involved in phytate hydrolysis and utilization, subcellular localization and biochemical properties of SgPAP23 will be characterized. Furthermore, to reveal the critical N-glycosylation sites of SgPAP23 function, the effects of the different N-glycosylation sites mutation on the activity and localization of SgPAP23 will be evaluated. Our results could not only dissect the function and regulation mechanisms of SgPAP23 in stylo adaptation to P deficiency, but also provide theoretical base and candidate gene for breeding forage cultivar with high P efficiency.

柱花草是热带和亚热带地区重要的豆科牧草。在低磷环境中，柱花草能高效利用土壤中的植酸磷，但其潜在的分子机理仍不清楚。植物紫色酸性磷酸酶（PAP）在活化水解植酸磷过程中发挥着重要作用。前期工作中鉴定到1个低磷特异诱导的柱花草紫色酸性磷酸酶SgPAP23，超量表达SgPAP23能显著提高转基因拟南芥对植酸磷的利用能力。本项目在此基础上，利用Real-time PCR和Western-blot等技术，在转录和蛋白水平上，分析SgPAP23对低磷胁迫的应答模式；通过研究SgPAP23的亚细胞定位、体外和体内生化酶学特征，阐明SgPAP23活化植酸磷的生物学功能；通过分析不同N-糖基化位点突变对SgPAP23酶活性和蛋白定位的影响，鉴定调控SgPAP23功能的关键N-糖基化修饰位点。研究结果将解析柱花草SgPAP23在缺磷适应性中的功能和调控机理，并为选育磷高效牧草品种提供理论依据和候选基因资源。

柱花草是重要的热带豆科牧草。在酸性缺磷土壤中，柱花草能高效利用植酸磷，但潜在的分子机理仍不清楚。前期研究中，我们从柱花草根系克隆了1个可能参与植酸磷利用的候选基因—紫色酸性磷酸酶（PAP）家族基因SgPAP23。本项目以解析SgPAP23的生物功能为目标，主要的研究内容和结果如下：（1）通过转录组学和蛋白组学技术，分析了柱花草PAPs家族成员对低磷胁迫的应答模式，其中SgPAP23在转录和蛋白水平均受低磷处理显著上调表达；（2）通过亚细胞定位分析，确定了SgPAP23主要定位于细胞质膜；（3）通过生化酶学性质分析，确定了SgPAP23具有较高的植酸酶活性，最适pH为5，最适温度为30℃；（4）通过蛋白质谱技术，鉴定了SgPAP23在N282位点存在的N-糖基化修饰；（5）通过菜豆毛根与拟南芥转基因体系，开展了SgPAP23的功能验证，超量表达SgPAP23能显著增加转基因株系对外源植酸磷的利用能力。综上所述，我们的研究结果揭示了柱花草高效活化利用植酸磷的分子机理，并证明了SgPAP23是柱花草耐低磷性状的重要基因，这将为选育磷高效牧草新品种提供理论依据及基因资源。