Detection And Evaluation Of Gmsos1 Mutant Of Salt Tolerance In Soybean

Soybean(Glycine max)is an important crop which show moderately salt-tolerance.It is becoming more and more urgent to cultivate high salt-tolerant soybean germplasm resources by modern breeding methods which can grow well on marginal land such as saline-alkali land.Toxicity of Na+ is the main damage of salt stress to plants.SOS1(salt overly sensitive 1)is a plasma membrane Na+/H+ antiporter which can be phosphorylated by SOS2/SOS3 complex.The activated Na+/H+ antiporter promote Na+efflux or Na compartment to reduce damage to cells and keep salt resistance.In this study,the CRISPR/Cas9 gene editing has been used successfully to obtain the constitutively activated soybean Na+/H+ antiporter GmSOS1 mutants.In order to elucidate the correlation and regulation mechanism between GmSOS1 and salt tolerance,PCR and CAPS applied to identify T3 and T4 generation soybean mutants and classify them according to their mutation types.Under NaCl stress,mutants were classified into tolerant,moderate tolerant and sensitive types according to their phenotype and biomass.qPCR was used to detect the expression patterns of SOS1 genes in wild type and mutants.The results could indicate the relationship between salt tolerance and different mutation sites.Furthermore,the salt tolerance regulation mechanism of soybean GmSOS1 can be clarified.The mutants could be a valuable germplasm resources of salt-tolerant soybean.The main results of this study are as follows:(1)CRISPR/Cas9 system was applied to edit the linker between auto inhibitory domain and cyclic nucleotide binding domain of GmSOS1 in soybean.According to the analysis of multiple sequence alignment,soybean Gmsosl materials can be classified into the following five types:Gmsosl-1,Gmsosl-2,Gmsosl-3,Gmsosl-4,andGmsosl-5.Mutant Gmsosl-1 has an tryptophan codon and premature termination codon insertion at position 974 and 975.Mutant Gmsosl-2 has a P974S substitution and premature termination codon at 975.Mutant Gmsosl-3 has a P974V substitution and a isoleucine insertion at position 975.The DNA sequence of the mutant Gmsosl-4 had 22 nucleotides deletion which introduce a premature termination codon.Mutant Gmsosl-5 was alanine deletion at position 973.(2)The mutants Gmsosl-1,Gmsosl-2,Gmsosl-3,Gmsosl-4,and Gmsosl-5 were cultured with Hoagland solution which m supplemented with the indicated amounts of NaCl.According to the survival rate and the Na+/K ratio in leaves and roots under 125mmol/L NaCl solution stress,mutants can be classified as:mutants Gmsosl-3 and Gmsos1-4 show as highly salt-tolerance,mutant Gmsosl-2 show moderately salt-tolerance and mutant show salt-sensitive.The Gmsosl-5 was moderately salt-tolerant at low concentration NaCl solution and highly salt-tolerant in high concentration NaCl solution.(3)The expression level of SOS1 in root was highest in leaf,hypercotyl hypocotyl and root of 10-day-old seedlings.The expression of SOS1 in the leaf,hypercotyl,hypocotyl and root tissues of gmsosl mutants was similar to wild type,and there was no significant difference in the expression of SOS1 in different gmsos1.