Establishment Of Potato Genetic Transformation System And The Construction Of Gene Editing Vector Of Immune-related Gene StCAD7

As one of the most important food crops in China and the world,Potato（Solanum tuberosum L）has been affected by pathogenic bacteria for a long history.The yield and quality of potato is seriously threatened by late blight which is caused by Phytophthora infestans.Owing to the virulence variation and loss of resistance,it is of great significance to explore and utilize new resistance pathways.Previous studies in our lab showed that plant Cinnamyl Alcohol Dehydrogenase 7（CAD7）subfamily proteins negatively regulate plant resistance to Phytophthora,and its immune function is conserved in Arabidopsis thaliana and Nicotiana benthamiana.The CRISPR/Cas9 gene-editing technology is the third generation of genome-editing technology developed after ZFN and TALEN.Since 2013,CRISPR/Cas9 has been successfully applied in several plant species,including the Solanaceae family.In this study,we aim to establish a genetic transformation system suitable for potato Atlantic and Désirée,and obtain potato gene edited plants for StCAD7 gene and StPDS gene respectively using CRISPR/Cas9-mediated gene editing technology.1.Exploring the optimal hormone ratio for potato callus induction and differentiation:for the stems of Atlantic and Désirée,the optimal medium for callus induction is MS30+NAA（2mg/L）+6-BA（1mg/L）+KT（0.5mg/L）+2.4-D（1mg/L）+Van（200mg/L）+Cef（200mg/L）;the optimal medium for callus differentiation is MS20+ZT（1mg/L）+Van（200mg/L）+Cef（200mg/L）,and the frequency of adventitious bud induction was above90%.For the leaves of Désirée,the optimal callus induction medium is MG+NAA（5mg/L）+6-BA（0.1mg/L）+Van（200mg/L）+Cef（200mg/L）;The optimal medium for callus differentiation is MG+NAA（0.02mg/L）+ZT（2mg/L）+GA3（0.2mg/L）+Van（200mg/L）+Cef（200mg/L）,and the frequency of adventitious bud induction was 60%.2.Through comparison,we found that the optimal infection conditions of Agrobacterium GV3101 to potato stem segment were as follows:OD₆₀₀=0.5,infection time is 5min,co-culture time is 1.5-2d;The optimal antibiotic combination is Van（200mg/L）+Cef（200mg/L）.3.The research cloned the homologous genes of PDS and CAD7 as well as the promoter of StPDS from potato.According to the conserved sequence of sequencing results,we designed three target sites for StCAD7,and constructed eight CRISPR vectors,besides that,five target sites were designed for StPDS,and six vectors were constructed.4.Exploring the optimal conditions for genetic transformation in potato:Compared with CRISPR vector p KI1.1R（plasmid#85808）,psg R-Cas9-p CXSN was more suitable for potato genetic transformation.The transformation efficiency of Désirée was slightly higher than that of Atlantic;At the same time,the genetic transformation test with StPDS as the target gene did not obtain the transformed strain,while the genetic transformation test with StCAD7 as the target gene could obtain the normal transformed strain.