Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content  

作　　者：Haiying Xiang Binhuan Chen Shuo Wang Wanli Zeng Jiarui Jiang Weisong Kong Haitao Huang Qili Mi Shuang Ni Qian Gao Zhenghe Li 

机构地区：[1]Yunnan Academy of Tobacco Science,Kunming 650106,China [2]State Key Laboratory of Rice Biology,Institute of Biotechnology,Zhejiang University,Hangzhou 310058,China [3]Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests,Zhejiang University,Hangzhou 310058,China [4]Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province,Zhejiang University,Hangzhou 310058,China

出　　处：《aBIOTECH》2024年第4期449-464,共16页生物技术通报(英文版)

基　　金：supported by a grant from the Natural Science Foundation of Zhejiang Province(No.LZ20C140004);FCTC grants No.110202201009(JY-09)and 110202101034(JY-11);FYCTIC Grant No.2022JY03.

摘　　要：Tobacco(Nicotiana tabacum)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 berberine bridge enzyme-like protein(BBL)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 BBL genes.Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels,while other BBL 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.

关 键 词：Eggplant mottled dwarf virus Rhabdovirus Delivery CRISPR/Cas Genome editing Berberine bridge enzyme-like TOBACCO Nicotine Alkaloid 

分 类 号：S572[农业科学—烟草工业]