CRISPR/Cas9 Revolutionizes Macleaya cordata Breeding: A Leap in Sanguinarine Biosynthesis

Sanguinarine in Macleaya cordata is an important natural source substitute for feed antibiotics. The analysis of sanguinarine biosynthesis pathway is the basis for the improvement of M. cordata varieties. In this study, an efficient CRISPR/Cas9 gene knockout system for medicinal plant M. cordata was reported for the first time, and the target editing efficiency was 86.76 %. On this basis, we successfully knocked out the key gene McSMT of chelerythrine branch in the sanguinarine metabolic pathway, and obtained the gene knockout plants. This is the first time to verify the gene function of McSMT in M. cordata, successfully truncate the chelerythrine branch pathway, reconstruct the sanguinarine metabolic pathway in M. cordata, and significantly increase the content of sanguinarine branch pathway compounds, especially dihydrosanguinarine and sanguinarine, the average content increased by 4.95 times and 3.29 times, respectively. Finally, through transcriptome analysis, we found that in the McSMT knockout plants, the expression levels of genes in the isoquinoline alkaloid biosynthesis were up-regulated except for the McSMT gene. In addition, transcriptome sequencing also predicted some transcription factors with significant expression differences, which laid a foundation for further functional gene research and molecular breeding research.