Gene Editing And Functional Analysis Of Related To Salt Tolerance Of BpSOS1 In Betula Platyphylla Suk.

It is an effective strategy to apply the forest ecosystem to restore the increasing saline-alkali land area by biological means with the frequent occurrence of extreme weather and the irrational use of land have led to the increasing area of saline-alkali land in the world.As a pioneer tree species in Northeast China,birch(Betula platyphylla Suk.)has natural advantages in ecological restoration of difficult sites,meanwhile with high ecological value and economical value.Some studies have shown that birch is sensitive to salt.Therefore,studying the physiological mechanism of birch under salt stress can enrich the theoretical basis of birch under salt stress and provide a good basis for the cultivation of salt-resistant birch a in the future.The plasma membrane Na~+/H~+antiporter SOS1(Salt Overly Sensitive 1)is an Na~+efflux transporter located on plasma membrane of plant cells,which can alleviate the toxicity of Na~+to plant cells by Na~+efflux under salt stress.The function of SOS1 has been elucidated in Arabidopsis,rice,soybean and so on,but less in woody plants.Here,we generate sos1mutants by CRISPR/Cas9,and then screen the growth phenotype and the response under salt stress,to explore the role of the BpSOS1 gene in the salt resistance of birch.The results will enrich the function of SOS1 in woody plants and provide a foundation for further research on molecular breeding of salt-tolerant birch.The results obtained in this study are as follows:1.Bioinformatics analysis shows that there is one copy of BpSOS1(Bplat＿h1＿08G01891.1)in Betula platyphylla Suk.The CDS region of BpSOS1 gene is 3402 bp in length and encodes1133 amino acids,which are mainly distributed on the cell membrane.The gene structure showed there are 23 exons of BpSOS1.The protein similarity between BpSOS1 and At SOS1 is65.57%,there are four conserved functional domains.BpSOS1 was significantly induced in roots and under salt stress.2.There are two sg RNAs,sg RNA1 and sg RNA2 were designed on the 15th and 16th exons of the BpSOS1.Two vectors were constructed to target the BpSOS1 gene.The genetic transformation of mature zygote embryos of birch was carried out by Agrobacterium tumefaciens.A total of 492 explants were transformed,and 48 of them were transformed into foreign genes successfully.Totally,there are 18 mutants obtained.The genetic transformation rate was 9.76%,the rate of gene editing was 37.50%.3.A homozygous mutant bpsos1-i26 with mutation of g4406del57/p530del27 was obtained from T0 generation.A biallelic mutant(biallele),bpsos1-i24,with mutation of g4420del1/p L552*,g4413del4/p L551*,g4413del13/p I548*was identified.And there are 16heterozygous mutants were obtained.4.The root length of bpsos1 mutant were significantly shorter than those of the wild type.The plant height did not show significant difference with wild type.Under 50 m M Na Cl treatment,the Na~+efflux velocity of root was significantly lower than that of the wild type,and the K~+absorption velocity was significantly lower than that of the wild type.All mutants show salt sensitive under Na Cl treatment.All results suggest that a putative plasma membrane Na~+/H~+antiporter BpSOS1 is critical for salt stress tolerance in birch.