The Research Of CRISPR/Cas9-mediated Targeted Mutagenesis Of PDS Gene In Poplar And Tobacco

CRISPR/Cas system as a new gene editing tool,is now widely research and application,have been reported implementedgene knockout of specific sites in many species,such as,Mice,Arabidopsis,Tobacco,Zebrafish.But theusing of this system in the plant,especially the perennial woody plants were poor.Eight hydrogen lycopene dehydrogenase(PDS),is one of the key enzymes of carotenoids synthetic route,which can translate colorless lycopene into non-ferrous carotenoids.Presently,the functions of the PDS have been extensive researched,and the corresponding nucleic acids and proteins sequences of PDS in some species like bacteria,algae and higher plants have been identified.Key laboratory of southwest university of transgenic plants and safety control used the gene editing techniques to fixed-point knockout experiments for poplar PDS.The research of poplar and tobacco gene functions and their genetic transformation systems is the main research content of our laboratory.In this stud,we designed the carrier of CRISPR to knock out PDS gene in poplar using gene editing technology.The main results are following:1.According to accession number: AF360196 from Arabidopsis thaliana database,we obtained the poplar and Arabidopsis thaliana PDS gene sequences and selected genes as the object of this experiment to knock out which have the closest relation with Arabidopsis thaliana genes by analysis of blasting the protein sequences and phylogenetic relationships.2.Analyzed the exon and intron which would be knocked out,meanwhile selected the target spot which was blasted with the poplar genome data(Populus trichocarpa taxid: 3694)from the NCBI web site.After evaluated the off-target effects of every target spot,we selected the lowest off-target effect one as our experimental object.3.We selected a high off-target effect spot which the first base is G without addition from literature about tobacco knockout.4.Designed the relative g RNA which was used for synthesizing the primers and built the sg RNA + Cas9 carrier which was detected with PCR and enzyme digestion to ensure its integrity and achievement.5.We transformed the recombined carrier to poplar and tobacco with agrobacterium and obtained transgenic plants.6.Shorter plant and leaf-albefaction are the main different phenotype between transgenic line and wild-type line.However,we failed to detect the carrier in poplar transgenic plants which may be on account of misoperation and inappropriate primers.