Fang Zhen, Zhang Kai, Li Jing, Ma Juan, Ye Chunxiu
College of Forestry and Landscape Architecture, Xinjiang Agricultural University, Urumqi, 830052, Xinjiang, China.
Mol Biotechnol. 2025 Jun;67(6):2319-2338. doi: 10.1007/s12033-024-01199-2. Epub 2024 Jun 2.
To investigate the biological effects of the ABA receptor pyrabactin resistance 1-like (PYR1-like) in Malus sieversii seeds, the proteins interacting with MsPYR1-like were screened by the membrane yeast two-hybrid library based on the split-ubiquitin system, and to construct the bait vector pBT3-SUC-PYR1 for Malus sieversii cDNA library, which had no self-activating effect on the yeast cells of the pPR3-N membrane yeast two-hybrid library. The library titer assay showed that it could meet the requirements for membrane yeast two-hybrid library screening. After sequencing, GenBank database blast, and yeast rotary validation, 28 candidate proteins interacting with MsPYR1-like were obtained, including ribosomal proteins, late embryogenesis abundant proteins, F-actin-capping proteins, phytochrome-interacting proteins, low-temperature-inducible 65 kDa protein-like, senescence-associated, PP2C and SnRK2 family members, and unknown proteins. Gene ontology analysis of the interaction proteins was related to plant hormone response and negative regulation of seed germination, overexpression of MsPYR1-like in Arabidopsis negatively regulates seed germination, and the study of the biological roles of MsPYR1-like interacting proteins lays the foundation for revealing the lifting of seed dormancy in Malus sieversii.
为了研究脱落酸（ABA）受体类吡咯啉酮抗性蛋白1（PYR1-like）在新疆野苹果种子中的生物学效应，基于分裂泛素系统，利用膜酵母双杂交文库筛选与MsPYR1-like相互作用的蛋白质，并构建对pPR3-N膜酵母双杂交文库的酵母细胞无自激活作用的新疆野苹果cDNA文库诱饵载体pBT3-SUC-PYR1。文库滴度测定表明其满足膜酵母双杂交文库筛选要求。经测序、GenBank数据库比对及酵母回转验证，获得了28个与MsPYR1-like相互作用的候选蛋白，包括核糖体蛋白、晚期胚胎丰富蛋白、F-肌动蛋白封端蛋白、光敏色素相互作用蛋白、低温诱导65 kDa类蛋白、衰老相关蛋白、PP2C和SnRK2家族成员以及未知蛋白。对相互作用蛋白的基因本体分析与植物激素应答及种子萌发的负调控有关，在拟南芥中过表达MsPYR1-like会负调控种子萌发，对MsPYR1-like相互作用蛋白生物学作用的研究为揭示新疆野苹果种子休眠解除奠定了基础。
