基于等温滴定微量热技术的玉米脱落酸受体检测体系

脱落酸(ABA)是响应逆境胁迫及调控植物生长发育的重要激素,其受体的发现以及在不同植物中的比较研究具有重要的理论与实际意义。等温滴定微量热技术(ITC)是鉴定和筛选ABA受体的重要技术之一,该方法对受体蛋白的纯度和生物活性要求较高。该文探讨了超声波破碎条件对受体蛋白得率以及ABA和受体蛋白浓度对二者亲和力的影响。结果表明,通过超声波破碎获得的原核表达玉米(Zea mays)ABA受体蛋白Zm PYL1含量高,蛋白质图谱条带清晰。超声波破碎适宜的条件为:菌悬液浓度100 mg·m L–1,破碎总时长15分钟,单次破碎时长为3秒,间歇时长10秒;ITC检测结果发现,(±)-ABA与玉米受体Zm PYL1的结合反应为吸热过程,推测该受体蛋白Zm PYL1为二聚体,4 mmol·L–1(±)-ABA与0.1 mmol·L–1受体蛋白Zm PYL1反应结合效果较好,反应的解离常数Kd值为72.46μmol·L–1。研究结果为筛选和鉴定植物ABA受体奠定了重要技术基础。

Optimization of Detection Methods for Zea mays ABA Receptor ZmPYL1 by Isothermal Titration Calorimetry

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Identification of the ABA receptor has important theoretical and practical significance. Isothermal titration calorimetry (ITC) is one of the important techniques used to identify and select ABA receptors; however, high- quality receptor protein is needed in this method. In this study, we examined factors affecting the separation and purification of the Zea mays ABA receptor ZmPYL1 and the binding affinity of ZmPYL1 to ABA. ZmPYL1 was overexpressed in E. coli, and high-level ZmPYL1 was purified from sonicated E. coli cells. The optimized ultrasonification conditions were total time 15 min, single time 3 s, internal time 10 s, and concentration of bacterial suspension 100 mg·mL^-1. The binding affinity reaction of (±)-ABA to ZmPYL1 was an endothermic process, so ZmPYL1 is a dimer receptor. ITC results showed K_d of 72.46 μmol·L^-1 for the interaction between 4 mmol·L^-1 (±)-ABA and 0.1 mmol·L^-1 ZmPYL1. This study provides an important technical basis for screening and identifying plant ABA receptors.