Usman Babar, Zhao Neng, Nawaz Gul, Qin Baoxiang, Liu Fang, Liu Yaoguang, Li Rongbai
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530004, China.
State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Bioresources, South China Agricultural University, Guangzhou 510642, China.
Int J Mol Sci. 2021 Mar 22;22(6):3225. doi: 10.3390/ijms22063225.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the () gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas9）介导的基因组编辑已成为作物分子育种的重要途径。为促进水稻育种，我们编辑了（）基因以获得有价值且稳定的长粒水稻突变体。此外，采用基于相对和绝对定量等压标签（iTRAQ）的蛋白质组学方法来确定突变体与野生型（WT）相比全蛋白质组的变化。设计了两个靶位点来构建载体，并采用农杆菌介导法进行水稻转化。成功引入了特定突变，与野生型相比，突变体的粒长（GL）和千粒重（GWT）分别增加了31.39%和27.15%。基于iTRAQ的蛋白质组学分析表明，突变体中共有31种蛋白质差异表达，其中20种上调，11种下调。结果表明，差异表达蛋白（DEP）主要与半胱氨酸合酶、半胱氨酸蛋白酶抑制剂、液泡蛋白分选相关蛋白、泛素和DNA连接酶有关。此外，功能分析表明，DEP大多富集于细胞过程、代谢过程、结合、跨膜、结构和催化活性。通路富集分析表明，DEP主要参与脂质代谢和氧脂生物合成。蛋白质-蛋白质相互作用（PPI）网络发现，与DNA损伤结合、泛素-40S核糖体和半胱氨酸蛋白酶抑制剂相关的蛋白质显示出较高的相互作用程度。利用CRISPR/Cas9系统获得了具有稳定遗传和长粒表型的纯合突变体系。本研究提供了一种提高粮食产量的便捷有效方法，可显著加速长粒粳稻亲本的育种进程，促进高产水稻的发展。
