Pei Yakun, Cao Wenhan, Kong Xiangchenxi, Wang Shaokang, Sun Zhongjuan, Zuo Yayun, Hu Zhaonong
Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau of Ministry of Agriculture and Rural Affairs, College of Plant Protection, Northwest a&F University, Yangling, 712100, Shaanxi, China.
Key Laboratory for Botanical Pesticide R&d of Shaanxi Province, Yangling, 712100, Shaanxi, China.
Planta. 2025 Mar 3;261(4):73. doi: 10.1007/s00425-025-04657-6.
Combined with hairy root transformation, the CRISPR/Cas9 system was established to initiate targeted mutagenesis of PlCYP81Q38, which influenced lignan accumulation in Phryma leptostachya. Phryma leptostachya is a traditional Chinese medicinal herb renowned for its applications in both conventional medicine and natural botanical insecticides, with lignans as the main active ingredients. During the biosynthesis of lignans, PlCYP81Q38, a P450 protein, is assumed to play a crucial role and is accountable for the production of sesamin from (+)-pinoresinol. As a cutting-edge genome editing tool, the CRISPR/Cas9 system is widely employed across diverse species for gene functional research but yet to be harnessed in P. leptostachya. This study utilized the CRISPR/Cas9 system in conjunction with hairy root transformation to initiate targeted mutagenesis in PlCYP81Q38 gene. Employing binary vectors, pYLCRISPR/Cas9Pubi-H, complemented by dual single-stranded guided RNAs (sgRNAs), enabled precise editing at two gene sites and the deletion of large fragments. This editing system resulted in mutagenesis rates surpassing 79%, achieving a notable rate of 61.9% fragment deletion mutants. Liquid chromatography/tandem mass spectrometry confirmed the impact on lignan biosynthesis by PlCYP81Q38-targeted mutagenesis, resulting in the accumulation of pinoresinol and disrupted production of sesamin, 6-demethoxy-leptostachyol acetate, and leptostachyol acetate. Furthermore, the knockout of PlCYP81Q38 up-regulated its upstream pathway genes, such as dirigent gene, cinnamoyl-CoA reductase genes, cinnamyl-alcohol dehydrogenase genes, and p-coumarate 3-hydroxylase genes, identified through gene co-expression analysis. Collectively, mediated by the CRISPR/Cas9 platform, the new biotechnology for targeted genome editing within P. leptostachya, our findings affirm the significant roles of PlCYP81Q38 in the lignan biosynthesis pathway and highlight the potential of CRISPR/Cas9 in exploring the functional genome and secondary metabolite biosynthesis of this plant species.
结合毛状根转化技术，建立了CRISPR/Cas9系统，以引发对影响细叶水团花中木脂素积累的PlCYP81Q38进行靶向诱变。细叶水团花是一种传统中药材，在传统医学和天然植物杀虫剂中都有应用，木脂素是其主要活性成分。在木脂素的生物合成过程中，一种P450蛋白PlCYP81Q38被认为起着关键作用，负责从(+)-松脂醇生成芝麻素。作为一种前沿的基因组编辑工具，CRISPR/Cas9系统被广泛应用于不同物种的基因功能研究，但尚未在细叶水团花中得到应用。本研究利用CRISPR/Cas9系统结合毛状根转化技术，对PlCYP81Q38基因进行靶向诱变。采用二元载体pYLCRISPR/Cas9Pubi-H，并辅以双单链引导RNA（sgRNA），能够在两个基因位点进行精确编辑并删除大片段。该编辑系统的诱变率超过79%，片段缺失突变体的比例达到了显著的61.9%。液相色谱/串联质谱证实了PlCYP81Q38靶向诱变对木脂素生物合成的影响，导致松脂醇积累，芝麻素、6-去甲氧基-细叶水团花醇乙酸酯和细叶水团花醇乙酸酯的生成受到破坏。此外，通过基因共表达分析发现，PlCYP81Q38的敲除上调了其上游途径基因，如 dirigent基因、肉桂酰辅酶A还原酶基因、肉桂醇脱氢酶基因和对香豆酸3-羟化酶基因。总的来说，在CRISPR/Cas9平台介导下，细叶水团花中靶向基因组编辑的新生物技术，我们的研究结果证实了PlCYP81Q38在木脂素生物合成途径中的重要作用，并突出了CRISPR/Cas9在探索该植物物种功能基因组和次生代谢物生物合成方面的潜力。
