Functional Analysis Of Resistance To Acid Aluminum Of GsMYB10 In Wild Soybean(Glycine Soja)

MYB transcription factor is one of the largest families of transcription factors in plants and regulates the processes of plant growth and development involving the responses against biological and abiotic stresses,development,differentiation,metabolism,defense of plants,and so on.Glycine Soja holds abundant genetic diversity and possesses a variety of resistance gene resources.In this paper,the gene GsMYB10 was selected from the gene expression profile resistant to acid aluminum of Glycine Soja BW69 line for further study.The main results are shown as follows:Bioinformatics analysis showed that the cDNA length of GsMYB10 is 1245bp encoding 414 amino acid residues.The results of the protein structure prediction using the NCBI database indicated that the GsMYB10 protein is a member of the MYB transcription factor family belonging to the 1R type.GsMYB10 protein contains two conserved domains,the MYB-like DNA binding domain located at the sites of 50-106 amino acid residues and the MYB-CC transfactor domain is located at the sites of 154-199 amino acid residues.Using recombinant DNA technology,the coding sequence of GsMYB10 gene was inserted into pCAMBIA1302 to form fusional GsMYB10-pCAMBIA1302 vector mediated further by Agrobacterium GV3101 for transient transformation of tobacco injection.The results of fluorescence microscopic observation showed that the GsMYB10 protein was localized in the nucleus of tobacco cell.Moreover,the coding sequence of GsMYB10 gene was constructed on the pGBKT7 vector to be pGBKT7-GsMYB10 using the recombinant DNA technology which was then transformed into the yeast cell of Y2Hgold.The results showed that GsMYB10 protein has the characteristic of transcriptional autoactivation activity.Real-time RT-PCR was used to analyze the expression pattern of the tissues.The results showed that the expression of GsMYB10 gene was relatively high in flowers,pods,and roots,while the expression level of GsMYB10 was the lowest in the apical tissues.Under different conditions of acid-aluminum stress,the expression of GsMYB10 gene was up-regulated by acid-aluminium stress.The expression trend of GsMYB10 gene first showed a significant increase and then slowly decreased with the increase of aluminum concentration.The highest expression level was at the concentration of 50?M AlCl₃?pH4.5?which was about 6 times compared to that of the control treatment.Time course testing of GsMYB10 gene to aluminum-aluminum stress showed that GsMYB10 gene was upregulated with a rapid increase and then followed by a slow decline with treatment time.The expression level of GsMYB10 reached the highest at the 8-hour time point of Al treatment about 5.9 times than that of the control treatment.The results of the expression pattern indicated that the GsMYB10 gene responds to the acid-aluminium stress.The GsMYB10 gene was transformed into the cultivar Huachun6 using the genetic transformation method of soybean cotyledonary-node mediated by Agrobacterium tumefaciens EHA01.All the transformants were obtained and propagated to the T₃generation.A total of 5 overexpression lines were obtained by the screening method of glufosinate,DNA level and RNA level identification.Using Huachun6 and three GsMYB10transgenic lines of T₃ generation for acid-aluminum stress treatment and hematoxylin staining,it was found that the overexpression of GsMYB10 gene enhanced the tolerance of the transgenic lines to acid-aluminum stress.Under the acid-aluminum stress,significant differences of test indexes were found between GsMYB10 transgenic lines and wild type,including the relative elongation ratio of the main root of the GsMYB10 transgenic line up to 50%,the aluminum content in the root tip of 60?g/g,the free proline content of 90?g/g,the root biomass of 46%and the malondialdehyde content of 7?mol/g in GsMYB10transgenic lines.