Functional Analysis Of Resistance To Acid Aluminum Of GsMYB7 In Wild Soybean(Glycine Soja)

Aluminum toxicity is one of the important factors limiting soybean growth and yield in acidic soil.The wild soybean of South China has more extensive genetic diversity than that of the cultivated soybean with the characteristic of strong adaptability to acid aluminum stress,among which many genes are involved in soybean resistance to aluminum toxicity.In this study,the up-regulated gene GsMYB7 was cloned according to the information the gene expression profile of wild soybean resistant to acidic aluminum,and then it was investigated for expression pattern,biochemical characteristics and the function of resistance to acidic aluminum.The main results are described as follows:The full-length of GsMYB7 was 2179 bp,which encodes 333 amino acid residues from the coding sequence of 1002 bp.Some response elements such as AE-box,ERE,TGA,HSE and TC rich repeats were discovered in the 1500 bp upstream region of GsMYB7which are involved in light,ethylene,auxin,heat and stress responses.Protein structure prediction showed that GsMYB7 protein holds the classical characteristic of R2R3 MYB transcription factor containing two conservative MYB domains which are located at the regions of 10-60 and 70-120 amino acid residues.The results of pHylogenetic analysis demonstrated that GsMYB7 was similar to the putative MYB protein from Arabidopsis thaliana.The coding sequence of GsMYB7 gene was inserted into the downstream of GFP sequence of pYL322-d1 vector,and then subcellular localization of GsMYB7 protein was carried out using Arabidopsis protoplasts.The results showed that GsMYB7 protein was localized in the nucleus of protoplasts.The GsMYB7 gene sequence was inserted between the restriction endonuclease sites of Nco I and BamHI of the pGBKT7 vector which was then transformed into yeast.The results indicateed that GsMYB7 protein has characteristic of transcriptional activation activity.AlCl₃ concentrations designed as 0,15,30,50,75,100?M?pH4.5?were carried out to treat the seedlings roots of wild soybean BW69 line.Quantitative RT-PCR results showed that GsMYB7 was up-regulated in roots of wild soybean which is up to the highest expression level at the AlCl₃ concentration of 75?M.After the DNA sequence containing 35S-GsMYB7-NOS was inserted into restriction endonuclease site of HindIII at multiple cloning sites of p ZY101 vector,the expression vector of GsMYB7-pZY101 was transformed into soybean Huachun 6 using the method of agrobacterium mediated soybean cotyledonary node for genetic transformation to obtain the transformants.Seven overexpression transgenic lines of Gs MYB7 were gained by the reproduction and molecular identification at DNA and RNA levels.Huachun 6 and transgenic overexpression lines of GsMYB7 from T4 generation were treated at the condition of acid aluminum to evaluate the resistance to Al stress.The results show that the relative root elongation,relative total root length,relative total root surface area and relative root volume of GsMYB7 transgenic lines were significantly higher than those of wild type.Under the condition of 30?M AlCl₃ treatment,the relative expressions of GsMYB7 gene in the transgenic lines were up to the highest about twice level than those in wild type.