Wen Dong, Wu Lan, Wang Mengyue, Yang Wei, Wang Xingwen, Ma Wei, Sun Wei, Chen Shilin, Xiang Li, Shi Yuhua
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Artemisinin Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
College of Pharmaceutical Sciences, Heilongjiang University of Chinese Medicine, Harbin, China.
Front Plant Sci. 2022 May 12;13:879390. doi: 10.3389/fpls.2022.879390. eCollection 2022.
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is an efficient genome editing tool used in multiple plant species. However, it has not been applied to Tartary buckwheat (), which is an important edible and medicinal crop rich in rutin and other flavonoids. is an R2R3-type MYB transcription factor that negatively regulates flavonoid biosynthesis in Tartary buckwheat. Here, the CRISPR/Cas9 system polycistronic tRNA-sgRNA (PTG)/Cas9 was employed to knock out the gene in Tartary buckwheat. Two single-guide RNAs (sgRNAs) were designed to target the second exon of the gene. Twelve transgenic hairy roots were obtained using mediated transformation. Sequencing data revealed that six lines containing six types of mutations at the predicted double-stranded break site were generated using sgRNA1. The mutation frequency reached 50%. A liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ-MS) based metabolomic analysis revealed that the content of rutin, catechin, and other flavonoids was increased in hairy root mutants compared with that of lines transformed with the empty vector. Thus, CRISPR/Cas9-mediated targeted mutagenesis of effectively increased the flavonoids content of Tartary buckwheat. This finding demonstrated that the CRISPR/Cas9 system is an efficient tool for precise genome editing in Tartary buckwheat and lays the foundation for gene function research and quality improvement in Tartary buckwheat.
成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）技术是一种应用于多种植物物种的高效基因组编辑工具。然而，它尚未应用于苦荞，苦荞是一种富含芦丁和其他黄酮类化合物的重要食用和药用作物。FtMYB11是一种R2R3型MYB转录因子，对苦荞中黄酮类化合物的生物合成起负调控作用。在此，采用CRISPR/Cas9系统多顺反子tRNA-sgRNA（PTG）/Cas9敲除苦荞中的FtMYB11基因。设计了两个单向导RNA（sgRNA）靶向FtMYB11基因的第二个外显子。通过介导转化获得了12条转基因毛状根。测序数据显示，使用sgRNA1产生了6个品系，在预测的双链断裂位点含有6种类型的突变。突变频率达到50%。基于液相色谱-串联四极杆质谱（LC-QqQ-MS）的代谢组学分析表明，与用空载体转化的品系相比，毛状根突变体中芦丁、儿茶素和其他黄酮类化合物的含量增加。因此，CRISPR/Cas9介导的FtMYB11靶向诱变有效地提高了苦荞中黄酮类化合物的含量。这一发现表明，CRISPR/Cas9系统是苦荞精确基因组编辑的有效工具，为苦荞基因功能研究和品质改良奠定了基础。
