Abstract
Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.
Highlights
Plants are constantly exposed to diverse and changing environmental conditions
The N-terminal domains are responsible for binding with the mRNA transcript, and the C-terminal region is responsible for protein-protein interaction during the splicing process
Our study provides excellent resource materials to further analyze the biological function of a family of splicing regulators in rice growth, development, and stress responses
Summary
Plants are constantly exposed to diverse and changing environmental conditions. Changes in their habitat can adversely affect their growth and development [1,2]. Plants have evolved numerous molecular mechanisms to survive under environmental fluctuations. Regulation of gene expression is a major form of adaptation in response to biotic and abiotic stresses [3,4]. Pre-mRNA splicing is primarily a co-transcriptional process conserved among eukaryotes. It is a key step in gene regulation and is performed by the spliceosome, a large macromolecular complex
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