利用CRISPR/Cas9系统定点敲除元宝枫AtruPDS基因的研究

Acer truncatum is a unique tree species in China,with ornamental,medicinal,and edible values.Its gene editing research has not meant reported yet.To establish a gene editing system for Acer truncatum,a regeneration system was established using embryos as explants.The phytoene dehydrogenase gene(AtruPDS)of Acer truncatum was used as the target gene.Designed sgRNAs at the first exon and 3'end,and pCambia1300-psgR-Cas9-sgRNA(target 1)-pAtU6-sgRNA(target 2)was constructed.Using Agrobacterium-mediated transformation of Acer truncatum embryos for gene editing.The results showed that the optimal formula for inducing adventitious buds from Acer truncatum embryos was DKW+ZT 0.05 mg/L+6-BA 0.25 mg/L+NAA 0.1 mg/L+sucrose 30 g/L+agar powder 8 g/L,pH 5.8;preliminary screening by PCR and phenotype detection resulted in gene edited positive seedlings.In summary,this study established a regeneration system using Acer truncatum embryos as explants and achieved targeted knockout of the AtruPDS gene using CRISPR/Cas9 technology,laying the foundation for functional genomics research and germplasm innovation of Acer truncatum.