Using Genetic Transformation And CRISPR/Cas9 Technology To Study The Function Of Soybean Mosaic Virus Resistance Gene GmRsc7-1

Soybean（Glycine max（L.）Merr.）is rich in protein and oil,and is an important oil crop and protein source in China.Soybean mosaic virus（SMV）seriously harms soybean growth and development,cuasing yield loss and destroying seed quality.The main soybean producing areas across the country are all threatened by different SMV strains,among which the SC7 strain is prevalent in the Huanghuaihai region.Through genome-wide association analysis,SC7 resistance-related sites have been excavated,and the candidate gene Gm Rsc7-1 has been determined.According to the results of sequence Blast analysis,Rsc7-1 is homologous to ANXUR1,ANXUR2 and FER in Arabidopsis.They belong to the malectin-like receptor kinase family and are new types of resistance gene.The CRISPR/Cas9 system is currently the most widely used gene editing tool,and its accuracy in targeting and editing endogenous genes has gradually improved.In this study,a multiple knockout vector p SC-M-RSC7 targeting Gm Rsc7-1 were constructed using the CRISPR/Cas9 multiple knockout vector modified by our laboratoryand an overexpression vector OE-Rsc7-1-3flag were constructed too.The targeting efficiency of the knockout vector was preliminarily verified by transformation of soybean hairy roots,and the knockout lines and overexpression lines were obtained through stable genetic transformation of soybean cotyledon nodes,further verifying the biological function of Gm Rsc7-1 in soybeans.The main results are as follows:1.The p SC-M-RSC7 vector was used to transform Agrobacterium rhizogenes K599 to induce soybean hairy roots,and the statistical hairy root targeting efficiency was 23.8%.2.The p SC-M-RSC7 vector and the OE-Rsc7-1-3flag vector were transformed into Agrobacterium tumefaciens EHA105 for infecting the soybean cotyledon nodes.We obtained 8 knockout-positive seedlings and 9 overexpression-positive seedlings respectively.Sequencing revealed that the targeting efficiency of knockout-positive seedlings was 87.5%,which was higher than that of hairy roots.3.A total of 12 transgenic clean and edited plants was detected T₁ knockout lines,and a total of 3 plants survived and seeds were harvested.Comparing to WT plants,the expression level of Gm Rsc7-1 was significantly decreased in the knockout plants,while significantly up-regulated in two overexpression lines plants,indicating that the target gene was successfully knocked out and overexpressed,respectively.KO1 plants showed obvious mosaic symptoms,while OE1 and OE2 leaves had slightly mosaic symptoms at 21 days past inoculation.In the top no-inoculated leaves,the expression levels of SMV-CP gene in KO1 plant was significantly up-regulated,while the expression of SMV-CP gene in OE1and OE2 plants was significantly down-regulated related to WT plants.The reactive oxygen species was stained by NBT,results showed that there were more dark blue reactive oxygen bursts in OE1 and OE2 leaves,while less dark blue dots in KO1 plants related to WT plants.The above results indicating that Gm Rsc7-1 can positively regulate both resistance to SC7and reactive oxygen burst.4.The test results of resistance-related hormone content in knockout plants showed that the content of ACC and SA-2-O-β-Glucoside in KO1 mutant plants were significantly reduced.We speculated that Gm Rsc7-1 may participate in disease resistance by affecting the content of ethylene and salicylic acid.