Abstract: 【Objective】Using CRISPR/Cas9 technology,the tobacco CENH3 gene was edited,and mutant plants with CENH3 gene mutations were obtained. An effective genome editing technology system was established after the editing efficiency and mutation type of the editing vector were identified.【Methods】The CENH3 editing vector was designed and constructed according to tobacco CENH3 gene sequence. Through Agrobacterium-mediated transformation,transgenic tobacco plants were produced,and sequencing was used to identify the CENH3 mutation types and the target gene editing efficiency of the transgenic plants. Droplet digital PCR was used to screen low-copy mutant plants.【Results】Among the 11 transgenic tobacco plants randomly tested,6 were edited,and the editing efficiency was 54.5%.In the edited plants,target 1 was not edited,and target 2 had four mutation types. All six of the edited plants had low copy numbers,ranging from 0.3 to 1.2.【Conclusion】The fixed-point editing of tobacco CENH3 was successfully achieved and CRISPR/Cas9 mediated tobacco CENH3 genome editing system was established.

Key words: Tobacco, CENH3 gene, CRISPR/Cas9, Transgene, Genome editing