PDF(Pubmed)
Abstract：
Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.
摘要：
泛素化在响应各种环境压力的蛋白质合成的翻译后阶段调节信号通路中起着至关重要的作用。已经发现E3泛素连接酶通过赋予待降解的蛋白质特异性来最终控制各种细胞内活性。进行这项研究是为了确认U盒E3型泛素连接酶（PUB）基因对水稻（OryzasativaL.）生物胁迫的生物学和遗传功能。设计了OsPUB9基因特异性sgRNA，并通过农杆菌介导的转化开发了转化体。使用愈伤组织进行深度测序以确认T0植物的突变类型，并进行总共三个步骤以选择没有T-DNA插入的无效个体。在OsPUB9基因编辑的情况下，基因编辑产生了一个bp的插入，并且证实了通过插入胸腺嘧啶来创建早期终止密码子和多个开放阅读框（ORF）位点。据推测，泛素化功能也随着U-boxE3泛素连接酶蛋白质结构的变化而变化。用细菌性叶枯病接种OsPUB9基因编辑的无效品系，并最终证实具有与Jinbaek相似的抗性表型，一种抗细菌性枯萎病的品种。因此,假设来自OsPUB9基因的氨基酸序列发生了很大变化，导致与生物学机制相关的原始蛋白质功能丧失。全面来说,已证实，当OsPUB9基因的特定位点发生突变时，对细菌性叶枯病胁迫的抗性增强。
