Functional Analysis Of Soybean GmVIT1 Gene In Response To Ironand Mamganese Stress

Although iron and manganese are essential trace elements for plants,excessive content can cause plant toxicity.The vacuolar iron transporter participates in the process of vacuolar detoxification and plays an important role in the storage and transportation of iron and manganese.The vacuolar iron transporter（VIT）is involved in This process,and plays an important role in the storage and transport of iron and manganese.The GmVIT1 gene in soybean was cloned and identified in the previous study of our laboratory,and the preliminary function was verified.In this study,the pBI121-GmVIT1-GFP expression vector was constructed for subcellular localization;the GmVIT1 gene promoter was cloned in Heinong 51 soybean,the pBI121-GmVIT1pro::GUS plant expression vector was successfully constructed,and the GmVIT1 gene promoter was transformed into In the model crop Arabidopsis thaliana;Phenotypic analysis and related physiological indicators detection of GmVIT1 gene T₃generation Arabidopsis thaliana under high iron and high manganese stress,and further analysis of the function of GmVIT1 gene,the main research results are as follows:1.Construction and subcellular localization of pBI121-GmVIT1-GFP vectorThe stop codon-removed GmVIT1 gene was linked with the pBI121-GFP expression vector to construct the pBI121-GmVIT1-GFP vector,which was transferred to GV3101 and transiently transfected in onion epidermal cells to confirm that the GmVIT1 was located on the tonoplast.2.Cloning of GmVIT1 Promoter Fragment and Construction of Plant Expression VectorA 1240 bp long GmVIT1 promoter fragment was obtained,and the analysis showed that the promoter fragment contained multiple cis-acting elements related to hormones and abiotic stresses.The pBI121-GmVIT1pro::GUS plant expression vector was transformed into Arabidopsis thaliana.GUS staining showed that under normal conditions,a small blue area appeared in the leaves of Arabidopsis thaliana seedlings.Under high iron stress,the leaves of Arabidopsis thaliana seedlings were almost all It is blue,indicating that pro GmVIT1 is active in leaves.3.Screening and identification of offspring of Arabidopsis thaliana transgenic with GmVIT1 geneThe GmVIT1 transgenic Arabidopsis thaliana seeds were screened by Kn screening medium,and the resistant seedlings of the T₂and T₃generations were obtained;the resistant seedlings were identified by PCR,and 8 T₂lines（L1,L4,L6,L8,L10,L12,L14,L20）,and the semi-quantitative results showed that L1,L4,and L10 had the highest expression levels,and 3 lines（L1,L4,L10）of the T₃generation were obtained.4.Physiological indicators of GmVIT1 transgenic Arabidopsis thaliana under high iron and high manganese stressThe GmVIT1 gene transgenic Arabidopsis thaliana（L1,L4,L10）and wild-type Arabidopsis（WT）were subjected to normal treatment and high iron and high manganese stress treatments.Under high iron and high manganese stress,the degree of yellowing of transgenic Arabidopsis thaliana leaves was lower than that of WT,and the contents of chlorophyll and soluble sugar were significantly higher than those of WT,high iron reductase（FCR）activity was significantly lower than WT under high iron stress,but significantly higher than WT under high manganese stress,SOD,CAT,POD enzyme activities were significantly higher than WT.In WT,the enzyme activities of SOD,CAT and POD were significantly higher than those in WT.The results indicated that the GmVIT1 gene Arabidopsis thaliana was tolerant to high iron and high manganese stress,and it was speculated that the VIT gene might be related to the vacuolar detoxification function.