Targeted Quantification of SnRK2.6 Activation Loop Phosphorylation Status at Positions 171 and 175/176 in NIL-DOG1 and dog1-1 Dry Seeds.

Stable transgenic seeds expressing SnRK2.6 as an N-terminally tagged YFP fusion in two genetic backgrounds, NIL-DOG1 and dog1-1, were used as the starting biological material. The target protein was purified through immunoprecipitation from native dry seed crude extracts of both genotypes in three independent biological replicates. Samples were split and measured using both DDA and PRM-based methods. This assay aimed to investigate the influence of the dog1-1 mutation on the phosphorylation status of SnRK2.6 activation loops.

在NIL-DOG1与dog1-1两种遗传背景中，表达N端标记黄色荧光蛋白（YFP）融合形式SnRK2.6的稳定转基因种子作为本研究的起始生物学材料。通过免疫沉淀法，从两种基因型的天然干燥种子粗提物中纯化靶蛋白，共设置三次独立生物学重复。将样品均分后，分别采用数据依赖采集（DDA）与平行反应监测（PRM）两种方法进行检测。本实验旨在探究dog1-1突变对SnRK2.6激活环磷酸化状态的影响。