Xiang Haiying, Chen Binhuan, Wang Shuo, Zeng Wanli, Jiang Jiarui, Kong Weisong, Huang Haitao, Mi Qili, Ni Shuang, Gao Qian, Li Zhenghe
Yunnan Academy of Tobacco Science, Kunming, 650106 China.
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058 China.
aBIOTECH. 2024 Nov 22;5(4):449-464. doi: 10.1007/s42994-024-00188-y. eCollection 2024 Dec.
Tobacco () plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six () family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of genes. Alkaloid profiling revealed that lines homozygous for and mutations exhibited drastically reduced nicotine levels, while other members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.
The online version contains supplementary material available at 10.1007/s42994-024-00188-y.
烟草植株合成具有精神活性的吡啶生物碱尼古丁，这引发了人们越来越浓厚的兴趣，即通过基因组编辑使关键生物合成基因失活来降低尼古丁水平。尽管稳定转化介导的基因组编辑在烟草中有效，但其多倍体性质使基因的完全敲除以及转基因从编辑植株中的分离变得复杂。在本研究中，我们通过工程化的负链RNA弹状病毒载体递送CRISPR/Cas机制，随后通过组织培养再生突变植株，从而在烟草中开发了一种非转基因基因组编辑方法。利用这种方法，我们针对商业烟草品种“宏达”中参与吡啶生物碱生物合成最后步骤的六个基因进行诱变。我们生成了一组16个突变系，这些突变系在不同基因组合中是纯合突变的。生物碱谱分析表明，纯合和突变的品系尼古丁水平大幅降低，而其他基因成员在尼古丁合成中起次要作用。这些品系中尼古丁含量的下降伴随着降烟碱和可替宁水平的降低，但去甲烟碱及其衍生物麦斯明水平的升高。初步农艺评价确定了两个低尼古丁品系，它们在温室和田间条件下的生长表型与野生型植株相当。我们的工作为培育低尼古丁烟草提供了潜在有价值的遗传材料，并增进了我们对生物碱生物合成的理解。
在线版本包含可在10.1007/s42994-024-00188-y获取的补充材料。
