Evaluating On Salt Resistance And Researching On Mechanism Of EsABI5 Gene Of Siberian Wildrye (Elymus Sibiricus L.)

Siberian wildrye(Elymus sibiricus L.)is a salt tolerant and high quality forage,not only has large potency in saline-alkali management,but also is an important resistant gene source for Triticeae crop.ABA pathway is an important signal transduction pathway in response to salt stress,ABI5 gene is an important transcription factor in ABA signal transduction and plays an important role in plants' resistance to stress.In this study,20 wild Siberian wildrye germplasm resources were evaluated salt tolerance during germination and seedling stage,and selected one high salt tolerance germplasm E02,cloning and sequencing EsABI5 from it.Screened the optimal reference gene,and analyzed EsABI5 expression under NaCl and ABA treatment.And identified the function of EsABI5 in transgetic tobacco.The aim of this study was to elucidate the mechanism of EsABI5.It can provide high salt tolerant germplasm and theoretical basis for the study on salt tolerant regulation,molecular mechanism and breeding of siberian wildrye and its related plants of wheat family.The main results were as follows:1.The order of salt tolerance of 20 wild Siberian wildrye germplasm resources were as follows: E02,E41,E15,E45,E19,E23,E31,E38,E42,E26,E18,E36,E20,E43,E01,E40,E21,E33,E17 and E14,and one germplasm with high salt tolerance was obtained.Squared euclidean distance=5,salt tolerance divided into four types,strong,medium,weak and very weak.Among them,strong salt tolerant germplasm accounted for 5%,medium salt tolerant germplasm accounted for 35%,weak salt tolerant germplasm accounted for55%,and very weak salt tolerant germplasm accounted for 5%.The proportion of the germplasm with strong and very weak salt tolerance were small,indicating that Siberian wildrye was more salt tolerant plant.2.The first obtained ABI5 sequence from Siberian wildrye was designated as EsABI5(Gen Bank no.: MN607227.1).EsABI5 encoded a 389-amino acid protein,which was hydrophilic unstable protein,no signal peptide and TMHMM,nuclear-localized,with obvious coiled coil areas.The expected protein product was EsABI5 is most homologous to Hv ABI5(Hordeum vulgare),the conserved domains were b ZIP46,CBLZ and b ZIP1.EsABI5 had 10 functional interaction proteins with a credibility greater than 0.7,including CIPK22,CIPK29,SAPK3 etc.Overall,EsABI5 belonged to the basic leucine zipper domain(b ZIP)superfamily and was closely related to the ABA signialing pathway and played an important role in the adaptation of plants to salt stress.3.EsABI5 was regulated by NaCl and ABA,the mechanisms of regulation were as follows: The expression level of EsABI5 was up-regulated in both roots and leaves under NaCl stress,however,the extend of up-regulation in leaves was larger than that in roots.Under ABA treatment,EsABI5 in leaves was also up-regulated,while in roots were down-regulated.When ABA concentration was 50 ?mol/L,the extend of down-regulation in roots(41-folds)was more greater than up-regulation in leaves(7-folds),at this time,proline content was obvious higher than control and 250 mmol/L NaCl,root activity and superoxide dismutase activity were the highest,malondialdehyde content was the lowest.Therefore,the down-regulation of roots EsABI5 expression may be the key to ABA signal transduction pathway and may play a negative regulatory role.4.EsABI5 gene was transferred into tobacco to further analyze its function.The amplified coding region of EsABI5 gene was successfully constructed into the vector p ART-CAM.The tobacco leaf disc was infected by agrobacterium-mediated method,and the buds were induced to differentiate,and the positive buds were obtained.T1-generation transgenic tobacco and wild-type tobacco were salted,the degree of yellowing and wilting of transgenic plants was more serious than that of wild-type plants.All physiological indexes showed that transgenic plants were more sensitive to salt stress.Among the 15 genes related to ABA and MAPK pathways,11 genes had lower expression WT than OT,including PYL4,PYL9,ABI1,MEK1,MKK3,MPK7,CPK17,ABI2,SAPK3,MPK4,MPK17.The expression of SAPK2,MAPKKK17,SAPK9 and SAPK10 genes of WT were higher than OT.Under salt stress,15 genes of WT were down regulated except MPK17.10 genes of OT were down regulated except SAPK3,MKK3,MAPKKK17,MEK1 and CPK17.There are complex regulatory relationships between EsABI5 and these genes,over-expression EsABI5 affected the ABA and MAPK pathway.Therefore,EsABI5 was involved in anti-salt responses in ABA signal transduction pathway and MAPK cascade pathway,and played a negative regulatory role during salt stress.