Functional Analysis Of Tolerance To Acid Aluminum Of GsGIS3 In Wild Soybean (Glycine Soja)

Aluminum（Al）toxicity limits plant growth and has a major impact on the agricultural productivity in acidic soils.Zinc finger protein（ZFP）family is one of the largest transcription factor families in plants which plays multiple roles in plant development and abiotic stresses.In this study,GsGIS3 was isolated from Al-tolerant wild soybean gene expression profiles,and it was functionally characterized in further studies,the main results are as follows:The GsGIS3 cDNA sequence was obtained from the NCBI database under gene locus LOC100811796 and protein accession number of XP＿003536676.1.The open reading frame（ORF）of gene GIS3 was 852 bp in length.The predicted GsGIS3 protein comprises 283amino acids（AA）.The GsGIS3 protein contained one typical zinc finger structure.Laser confocal microscopic observations demonstrates that GsGIS3 is a nuclear protein.The quantitative real time polymerase chain reaction（qRT-PCR）was used to analysis the expression patterns of GsGIS3.It was found that the expression of GsGIS3 was much higher level in stem than in leaf and root,the expression level of GsGIS3 was higher in 2-4cm root segments than in 0-2 and 4-6 cm root segments.Compared to the control,different Altreatment concentrations could all significantly induce the upregulation of GsGIS3expression,with the increase of Alconcentration,the expression of GsGIS3 gene showed a trend of increasing significantly at first and then decreasing slowly,while the expression improved up to the highest level of 6-fold at the treatment of 50μM AlCl₃ compared with those detected under control condition.In addition,we analyzed the temporal expression pattern of GsGIS3 under acidic Altreatment over a period of 24 h;the results showed that the expression of GsGIS3 gene increased rapidly and then decreased slowly with time,and it increased up to the highest expression level of 5-fold at 6 h.To investigate the expression patterns of GsGIS3 response to other abiotic stresses,GsGIS3 was upregulated significantly under NaCl and GA3 treatment.The overexpression vector and RNAi vector were injected with Agrobacterium rhizogenes pathogenic strain K599 to induce the soybean hairy root.Homologous expression of GsGIS3 in soybean hairy roots was done using Hematoxylin staining.It was found that the staining of soybean hairy roots with the overexpression of GsGIS3 was the shallower and that it was the deeper in RNAi soybean hairy roots compared to control.Through heterologous transformation of Arabidopsis,we identified five homozygous transgenic lines.Under treatment of 100μM AlCl₃,the root length of wild-type plants was 3.8 cm long,while the root lengths of GsGIS3 transgenic lines ranged from 5.5 to 5.8 cm long,the transgenic lines showed better growth than wild-type plants in Alstress.Via the observation of the c hydroponic experiments,the results showed that GsGIS3 transgenic lines had the more total root surface and total root length compared to the control,and higher proline and lower MDA accumulation under concentrations of AlCl₃.Moreover,GsGIS3 expression in Arabidopsis altered the transcriptional activities of the Al-tolerance-related genes and/or GAs synthesis and GAs signaling related genes.Under Alstress,the expressions of AtALMT1,AtALS3,and AtGA2OX1 were up-regulated in GsGIS3 transgenic lines,and the expressions of AtGA3OX1and AtGID1 were down-regulated in GsGIS3 transgenic lines as compared to those in WT plants.