Constitutive Expression Of RXEG1 To Improve Soybean Resistance To Root Rot Disease And Gene Editing Of Soybean

Soybean[Glycine max（L.）Merr.]is a necessary resource of food and oil for human life,but soybean diseases seriously affect the yield and quality of soybean.Resistant varieties is the most effective way to control diseases.However,the traditional breeding process of resistant varieties is facing many problems,such as long breeding cycle,lack of resistance sources,and loss of disease resistance due to virulence variation of pathogens.Therefore,the use of disease resistance gene engineering is one of the relatively effective methods to solve these problems.In this study,three transgenic soybean lines resistant to Rhizoctonia solani were obtained by heterologous expressing the tobacco PRR gene RXEG1 in soybean by Agrobacterium mediated transformation.At the same time,an efficient gene editing system was established in soybean,and the susceptible gene was knocked out by CRISPR/Cas9 gene editing technology.The main research contents are as follows:1.Overexpression of tobacco pattern recognition receptor gene RXEG1 in soybean significantly enhanced soybean resistance to Rhizoctonia solaniRXEG1 is a pattern recognition receptor that specifically recognizes the Pathogen-Associated Molecular Patterns XEG1 in tobacco,and is a key factor in plant defense response.In order to improve the resistance of soybean to root rot,the stable expression of tobacco PRR gene RXEG1 in soybean was achieved by Agrobacterium mediated genetic transformation using Williams 82 soybean genotype as a donor.Three soybean transgenic lines stably expressing RXEG1 were obtained by applying glyphosate leaf painting,PCR and real-time quantitative PCR.Through hypocotyl inoculation experiment,it was found that the resistance of transgenic materials to Rhizoctonia solani was significantly increased compared with wild type.2.Establishment of soybean gene editing systemIn order to establish a soybean gene editing system,we first optimized the transformation of soybean hairy root system,and then used this system to verify the different gene editing systems in soybean.At the same time,we established a soybean CRISPR/Lbcpf1multi-gene editing system to delete large segments of chromosome.In addition,the soybean susceptible gene Gm TAP1 was used as the target,and was knocked out by CRISPR/Cas9technology through Agrobacterium mediated transformation.The main results are as follows:1)Establishment of rapid and efficient transformation system of soybean hairy roots.The transformation system was optimized from three aspects:germination time,type of co-culture medium and concentration of screening agent.The results showed that the cotyledons germinated for 1 day were infected and co-cultured with Agrobacterium tumefaciens in 1/2B5 medium,and 3 mg/L herbicide was added to the rooting medium.The transformation efficiency was the highest,reaching 69%.The whole process of the optimized transformation system only takes 16 days,and it can be widely used in different soybean genotypes.At the same time,the system can be used as a powerful tool for high-throughput functional analysis of soybean genes,which can be applied to analyze protein expression,subcellular localization,protein-protein interaction,and evaluation of gene editing efficiency.2)Establishment of soybean CRISPR/LbCpf1 multi-gene editing system.In this study,CRISPR/LbCpf1 vector was constructed by cr RNA array driven by soybean U6 promoter.The length of direct repeat（DR）and guide RNA were optimized.The results showed that the combination of 21nt DR and 22nt guide RNA showed the highest editing efficiency,and the editing efficiency was up to 91.7%.Furthermore,multiple DR and cr RNA were constructed into a multi-gene editing system.It was found that CRISPR/LbCpf1 could simultaneously edit up to eight targets,and the editing efficiency was 17.1%.In this study,we found that LbCpf1 has a preference for the first and last crRNA in the crRNA array.Compared with CRISPR/Cas9 system,CRISPR/LbCpf1 could produce editing in the range of-3^rd-41^rdbp at the far end of PAM site.The optimized CRISPR/LbCpf1 multi-gene editing system was used to edit soybean FAD2-A gene cluster.The results showed that CRISPR/LbCpf1 system successfully deleted small segments（<1KB）,large segments（～10KB）and super large segments（>10KB）in soybean,and the deletion efficiency was 12.2%,12.5%and 9.5%respectively.3)Knockout soybean Gm TAP1 gene by CRISPR/Cas9 gene editing tool.Five gene editing vectors containing different sg RNAs of susceptible gene Gm TAP1 as the target were constructed.Using the optimized transformation of soybean hairy root system,the sg RNA2was found to induce efficient editing.The transformed plants were obtained by Agrobacterium tumefaciens mediated stable transformation of soybean.Two Gm TAP1knockout homozygous mutants were obtained by PCR,T7EI digestion and sequencing analysis.On the basis of the above,a Transgenic-free Gm TAP1 knockout mutant was screened,which provided a new research material for soybean disease resistance breeding.