Cloning And Functional Characterization Of VIT1 Gene From Soybean(Glycine Max)

Iron is an essential nutrient supporting life activities and is toxic in excess,thus iron homeostasis is important in living organisms.Soybean is an important economic crop which is sensitive to iron.Vacuolar iron transporter(VIT)is involved in the transport of iron and manganese ion between vacuolar and cytoplasm to regulate metal homeostasis.The main function of VIT is iron transport.In this study,GmVIT1 was cloned from heinong51 soybean,plant expression vector pBI121-GmVIT1 was construct and transferred into model plant,tobacco and Arabidopsis thaliana.The function of GmVIT1 was preliminarily explored by means of bioinformatics,phenotypic observation under iron stress and detection of related physiological indicators.Research showed that GmVIT1 plays an important role in tolerance to iron stress and the specific results are as follows:1.GmVIT1 gene obtaine and bioinformatics analysisCDS of GmVIT1 gene is 660 bp and encodes 219 amino acids.The prediction of physicochemical properties showed that Gm VIT1 protein was an acid hydrophobic protein.Among the secondary structures of GmVIT1,?-helix(Hh)accounted for53.88%,random coil(Cc)accounted for 30.14%,elongation chain(Ee)accounted for10.50%,and Angle of Angle(Tt)accounted for 5.48%,respectively.The tertiary structure of Gm VIT1 was predicted.The phylogenetic analysis showed that GmVIT1 had the closest relationship with ApVIT1,followed by CcVIT1.2.Tissue expression pattern of GmVIT1 gene under iron stressThe expression sequence of GmVIT1 gene was leaves > stem > roots in soybean.Compared with normal conditions,the expression of GmVIT1 in soybean roots,stems and leaves was up-regulated under iron deficiency and iron stress.The expression of GmVIT1 significantly increased in soybean leaves under iron-deficient and iron-excess and in soybean stems under iron-excess.3.Construction and genetic transformation of pBI121-GmVIT1 expression vectorThe cloned GmVIT1 was linked to pBI121 with Km resistance.The pBI121-GmVIT1 was transferred into Agrobacterium,which infects tobacco andArabidopsis thaliana.A total of 34 tobacco seedlings and 40 Arabidopsis thaliana seedlings resistant to Km were screened.A total of 17 tobacco seedlings were identified to be transgenic positive;the expression levels of GmVIT1 gene in T11,T33 and T34 were found to be relatively high by qRT-PCR detection.4.Physiological indicators of GmVIT1 transgenic tobacco under iron stressThe punching leaves of GmVIT1 transgenic tobacco(T11,T33,T34)and wild-type tobacco(WT)were stressed by iron deficiency and iron excess with the control of normal.Under iron deficiency and iron excess,the yellowing was more obvious in WT leaves than that of three transgenic lines.The chlorophyll contents were significantly higher in transgenic lines than that of WT.The activity of SOD,POD,CAT and soluble protein content were significantly higher and MDA of was significantly lower in transgenic lines than that of WT under iron excess.The above studies indicate that the overexpression of GmVIT1 gene improved the tolerance to iron excess in tobacco.