RESUMEN
Alfalfa (Medicago sativa L.) is the most widely cultivated leguminous herb in the world. Its agricultural development has been restricted by various adverse environmental conditions, including water deficiency, high salinity, and low temperature. WRKY transcription factors (TFs) serve important roles in the regulation of plant development and stress responses. Research on the WRKY gene family has been reported for several species, but minimal information is available for alfalfa. In the present study, a total of 107 WRKY genes were identified in alfalfa and divided into 3 main groups. The classification, evolution, conserved motifs, and tissue expression were comprehensively analyzed. Meanwhile, 27 MsWRKY candidate genes that may be involved in abiotic stress were isolated through an analysis of gene expression profiles under different stresses, including cold, abscisic acid, drought, and salt treatments. Additionally, investigation of the cis-elements and potential biological functions of these genes further revealed that MsWRKY TFs may serve important roles in multiple stress resistance in alfalfa. This study provides an important foundation for future cloning and functional studies of WRKY genes in alfalfa.
RESUMEN
Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.