A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding.The gene of interest is transferred to the vector by molecular cloning technology.Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity,efficiency,and standardization.In this study,we developed a"pNC"vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants.These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning.We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes,including the study of gene ectopic expression,protein subcellular localization,protein-protein interaction,gene silencing(RNAi),virus-induced gene silencing,promoter activity,and CRISPR-Cas9-mediated genome editing.The"pNC"vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.