Rice (Oryza sativa L.) is one of the world's most important staple crops and a powerful model system for studying monocot species because of its relatively small genome, rich genomic resources, and a highly efficient transformation system. With the completion of rice genome sequencing, the challenge of the post-genomic era is to systematically analyze the functions of all rice genes. Gene knockout is a frequently used and effective strategy for achieving this goal. Thus, generation of large-scale mutants at the whole-genome level is of great value for both functional genomics and genetic improvement of rice. Traditionally, large numbers of mutants are produced by physical, chemical, or biological mutagenesis. Mutants created by these methods have made enormous contributions to basic plant research and crop improvement. T-DNA insertion (Jeon et al., 2000), TILLING (targeting-induced local lesions in genomes) (Till et al., 2007) and RNAi (RNA interference) (Wang et al., 2013) are the three most common methods of performing genetic studies. T-DNA insertion and TILLING are time-consuming and labor-intensive in generating genome-wide mutant libraries, because large mutagenized populations must be generated to ensure sufficient genome-wide coverage. In addition, the T-DNA insertions occur randomly and often in intergenic and noncoding regions, for TILLING mutants it is difficult to identify the targeted mutations for the observed phenotypes, and the RNAi method only reduces the expression of targeted genes rather than generating the knockout mutants. Recently, a simple and highly efficient genomic engineering tool, the CRISPR (Clustered Regularly Interspaced Palindromic Repeats)/Cas9 system, has been developed; this technology can create small insertions and deletions (indels) in specific target genes and has been applied to many organisms. Because it is an easy and convenient technique, some CRISPR/Cas9 mutant libraries have been developed for genomewide mutation screens in cultured eukaryotic cells (Shalem et al., 2015). However, no large-scale CRISPR/Cas9 mutant libraries have yet been generated in higher plants. Here, we report the construction of a high-throughput CRISPR/Cas9 mutant library in rice and demonstrate its application for identifying gene functions and its potential use for genetic improvement.
Concise syntheses of parvistemin A and diperezone are achieved using ring expansion of cyclobutenones and oxidative phenolic coupling under basic conditions.
A Ni-catalyzed ring-opening of α-hydroxycyclobutenones is reported herein. A remarkable ligand effect was observed during transformations following the ring-opening. The employment of PPh leads to the formation of 2-furanones 2 through a migration of an alkoxyl group, and 2-furanones 3 were generated through a migration of hydrogen in the presence of Xantphos, affording a divergent approach to 2-furanones bearing multiple functional groups.
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