Preliminary Research On Sugarcane CRISPR/Cas9 Gene Editing Technology

As my country’s main sugar crop,sugarcane provides a guarantee for my country’s cane sugar production.At the same time,it can produce a large amount of ethanol as fuel.It is an efficient and clean renewable bioenergy.With the acceleration of social production and the increase in energy demand,there is an urgent need for new varieties of high-quality sugarcane.However,sugarcane’s own genome is complex and belongs to allopolyploid plants.However,the traditional breeding cycle is long,slow to take effect,and various factors restrict the development of high-quality new varieties of sugarcane.The newly emerging CRISPR/Cas9 gene editing technology,with its simple design and operation,low cost,and high editing efficiency,has become a new boost for sugarcane research.It not only provides sufficient technical support for the research of sugarcane gene function,but also develops a broad new direction for the improvement and cultivation of new germplasm.This research aims to obtain sugarcane gene-edited plants through CRISPR/Cas9 technology and establish an preliminary research on CRISPR/Cas9 gene editing technology of sugarcane.The specific research results are as follows:(1)Taking the sugarcane variety NK into which the glyphosate-resistant gene EPSPS was transferred as the recipient material,based on its gene fragments,a dual-target gene editing vector NK-EPSPS-Cas9 was constructed.(2)Through Agrobacterium-mediated genetic transformation of embryogenic callus of sugarcane variety NK,resistant plants were screened and cultivated.After double verification of the screening marker Bar gene and target gene,32 PCR-positive plants were obtained.(3)The DNA of 32 PCR-positive plants was extracted for sequencing and Hi-Tom target mutation analysis,and 9 NK-EPSPS-Cas9 gene-edited plants with mutations in the target site were obtained,and the editing efficiency was 28.13%.(4)Analyzing the mutations of 9 NK-EPSPS-Cas9 gene-edited plants,4 of them were fully edited,EPSPS-1,EPSPS-3,EPSPS-4,EPSPS-9,and the homozygous rate was44.44%.A total of 5 deletion and 1 insertion mutation types appeared in the 9 gene-edited plants,namely 1D,2D,3D,4D,10 D,and 1I.Except EPSPS-1,8 of them all showed 2D mutations,appeared the most frequently,accounting for 88.89%.3 of the 9 gene-edited plants had mutations at both targets,namely EPSPS-3,EPSPS-4,EPSPS-9,and the remaining 6 plants only had mutations at the EPSPS-T1 target.(5)9 NK-EPSPS-Cas9 gene-edited plants were tested for glyphosate resistance,and three groups of control plants were established at the same time,namely: CPB-Cas9 empty vector transformed plants,ROC22 non-resistant plants,and NK glyphosate-resistant plants.The test results showed that 4 fully edited plants withered and died,which proved that the EPSPS gene in the sugarcane variety NK was successfully edited;and 3 of the 4fully edited plants had mutations at both targets,indicating the double The editing effect of target mutation is better.In summary,this study completed the editing of the EPSPS of the glyphosate-resistant gene in the sugarcane variety NK,and completed the preliminary study of the sugarcane CRISPR/Cas9 gene editing technology,and also for the subsequent study of the sugarcane gene function through the CRISPR/Cas9 technology The provision of technical support and the creation and improvement of high-quality new varieties of sugarcane have laid a theoretical foundation and launched a new direction.