Study On Establishment Of CRISPR/Cas9 Technology And Targeted Genome Editing On Sh1 Gene In Agropyron Mongolicum Keng

Agropyron mongolicum Keng,the excellent forage grasses and important resource of vegetation restoration in China,has great significance in the development of agriculture and animal husbandry and improvement of ecological environment.Seed shattering,which is essential for the propagation of their offspring in wild plants,is of significant adaptive importance under strong selective pressures.However,it is a major cause of yield loss in crops.Traditional breeding method were based on cross breeding,which was time-consuming and was relied on the experience of breeders.Random mutagenesis means need to screen mutants by consuming a lot of human and material resources.Genome site-directed mutagenesis techniques have huge advantage.Traditional site-directed mutagenesis techniques technical process complex and difficult to widely.A new technology,which relies on an RNA-guided nuclease,was re-engineered to efficiently edit genomes across a variety of eukaryotes and has become an important tool for genome editing in plants.Up to now,few of studies have explored to the application of CRISPR/Cas9 system as a new genome targeting modification technology in Agropyron mongolicum Keng.In this study,our work includes two parts.Firstly,we established the transient transforrmation system of Agropyron mongolicum Keng;secondly,we focused on the using of CRISPR/Cas9 technology to edit Sh1 gene in callus of Agropyron mongolicum Keng.Our main results are as follows:1 Construction of transient transformation system of Agropyron mongolicum KengBy orthogonal test,we analyzed the effects of concentrations of cellulose,macerozyme,and mannitiol as well as the time of enzyme digestion on the productivity and vitality of protoplast from Agropyron mongolicum Keng stem and leaf respectively.40%PEG-Ca2+ was used to transfer the green fluorescent protein to test the transformation efficiency.The results showed that the best condition of protoplast isolation of stem and leaf was cellulase-R10 1.5%,macerozyme R-10 0.75%,mannitiol 0.6mol/L and 5h of enzyme digestion.On this condition,the productivity of protoplast from stem and leaf was 5×106 and 6×106 respectively,vitality was 93%and 90%respectively,transformation efficiency was 43-61%,and 38-50%respectively.2 CRISPR/Cas9 expression vectiors construction and targeted sites choiceIn our study,Sh1 gene,which is related to seed shattering of plants,was chosen as ttargeted genes.Briefly,using pRGEB32 vector to construct three sgRNA system vectors(pRGEB32-Sh1-1,pRGEB32-Shl-2,pRGEB32-Shl-3).3 Mutation detectionIn our study,we chosen sequences containing common restriction enzyme sites as targeted sites.PCR-RE method was used to detect the mutation.It was showed that there were mutations in all three targeted sites.Sequencing showed that,CRISPR/Cas9 system induced mainly to point mutation(C-T replacement)and small deletion(1 or 5bp deletion),their frequencies are 20%and 80%respectively.In this study,the mutation percentages of Sh1-1 site is 8%,4%for Sh1-2 site,and 8%for Sh1-3.Taken together,we first succeeded in isolation of protoplast and construction of transient transformation system of Agropyron mongolicum Keng.Our results demonstrated that CRISPR/Cas9 system can be applied as a powerful tool for gene targeting and genome editing in Agropyron mongolicuum Keng.Selecting effective target sites at protoplast level can provid reliable experimental basis for bombardment transformation.