Mining Of Salt Tolerance Genes In Tartary Buckwheat And Functional Verification Of Candidate Genes

Fagopyrum tataricum not only has high nutritional value,but also has strong abiotic stress resistance.It has been paid more attention in the food industry,feed industry and pharmaceutical industry,and can be used as a high-quality material for mining stress resistance genes.Now,with the expansion of saline alkali land,cultivating salt tolerant varieties is one of the key steps to solve the problem of maximizing land use.In this study,268 Fagopyrum tataricum germplasms from different provinces were used as materials.The seeds were treated with NaCl to observe their response to stress at germination stage.Genome wide association analysis was performed using the total Subordinate Function Values of 196 materials(10 fold resequenced)and salt tolerance traits to preliminarily locate candidate regions related to salt tolerance traits.Then the candidate genes were screened by analyzing the expression of candidate genes and haplotype analysis.The salt resistance of Fagopyrum tataricum hairy roots and Arabidopsis thaliana was evaluated by constructing the over expression vector.This will provide a theoretical basis for further mining salt tolerance genes,improving salt tolerance mechanism and breeding salt tolerant varieties of Fagopyrum tataricum.The main results are as follows:1.The salt tolerance of all materials was evaluated by using the total Subordinate Function Values of three salt tolerance related characters.The results showed that with the increase of NaCl concentration,the inhibition effect on seed germination rate,radicle length and epicotyl length was more obvious;Among them,24 accessions with the total Subordinate Function Values>1.5 and 17 accessions with the total Subordinate Function Values<0.7,showed that 24 accessions were not sensitive to salt,and 17 accessions were sensitive to salt.2.The total Subordinate Function Values of 197 materials(which have been resequenced)were analyzed separately.It was found that the total Subordinate Function Values were mainly distributed between the upper quartile(1.29)and the lower quartile(0.92).The number of materials above the upper quartile and below the lower quartile was less,and the general trend of normal distribution.GWAS association analysis was performed with the total Subordinate Function Values and genotype data of 197 accessions,a peak appeared on chromosome 6(Ft6:18420318),and the phenotypic data of the two haplotypes were significantly different.The regions with-log₁₀^(P)>5 were considered as salt tolerance candidate regions,and the genes within 100 kb of the left and right wings of the highest point were selected as candidate genes.Through gene annotation,NaCl induced expression analysis and haplotype analysis,two candidate genes Ft Pin G0606594100.01 and Ft Pin G0606596500.01 were screened out,which were named Ft MGT2L and Ft2OG oxygenases 1.The Ft MGT2L gene starts from Ft6:18327628 and ends at Ft6:18332564 on chromosome 6,with a total length of 4936 bp and CDS length of1326 bp,encoding 441 amino acids.The Ft2OG oxygenases 1 gene starts from Ft:18413012 and ends at Ft:18417968 on chromosome 6,with a total length of 4956bp,CDS length of 999 bp,and encoding 332 amino acids.3.Tissue-specific analysis shows that Ft MGT2L responds significantly in leaves and stems,and Ft2OG oxygenases 1 has a higher expression in flowers and fruits.Subcellular localization of candidate genes was performed on the online platform.The results shows that Ft MGT2L and Ft2OG oxygenases 1 have the highest probability in the cytoplasm.Phylogenetic tree analysis showed that Ft MGT2L was closely related to Ft MGT2 of Arabidopsis thaliana,which was a Mg²⁺channel protein,and which could effectively improve the plant resistance to inversion.The enzyme gene family of Ft2OG oxygenases 1 plays an important role in maintaining cell homeostasis.However,there are few studies on most genes in this family,which need further verification.4.Two candidate genes were cloned and ligated into p CAMBIA1302overexpression vector.The salt tolerance of Fagopyrum tataricum hairy root and Arabidopsis thaliana was identified.(1)Effects of NaCl stress on the germination of ectopic expression Arabidopsis thaliana:NaCl stress inhibited the growth of Arabidopsis thaliana,resulting in short plant,light yellow leaves,short roots and no lateral root growth.The germination rate(about 70%)of Ft MGT2L ectopic expression plants was higher than that of the control(62%),and there was no significant difference in root length and seedling length.The root growth of Ft2OG oxygenases 1ectopic expression plants was less affected by stress than that of wild-type plants.(2)Most of the rosette leaves in the lower part of wild-type Arabidopsis seedlings were withered after one week of salt stress treatment,and the damage degree was the largest.The leaves of overexpression plants were normal,dark green and a few withered and yellow.The results of DAB staining showed that the leaves of wild-type plants were dyed red in large area,the leaves of 1302 empty plants were red in part,and the over expression lines were spotted red in small area.In Arabidopsis thaliana with ectopic expression of Ft MGT2L,the activities of SOD and cat increased significantly under salt stress.The increase of SOD activity in Arabidopsis thaliana with ectopic expression of Ft2OG oxygenases 1 was also significantly higher than that in the control.This result is similar to that of overexpression of hairy roots.