Study On Regulation Of BHLH30 Transcription Factor From Tampa Black Soybean On GmSTOP1 And GmMATE75 Gene To Aluminum Stress Tolerance

The acid soil in China is mainly distributed in Southern area,and the proportion and acidification of the soil have been increasing.Aluminum toxicity is the major limiting factor in acid soil.In acidic conditions,especially when pH<5.0,aluminum is distributed in the soil in active ion form Al³⁺.Al³⁺can reduce the absorption of water and nutrients of plants,inhibit the elongation of the roots,and affect the growth and development of plants.In recent years,although there are many researches about aluminum stress and aluminum tolerance mechanism,the field is still at the stage of theory and exploration,and needs more support from theory and practice.Our previous research found that the bHLH30 transcription factor gene from Tampa black soybean was regulated by aluminum stress.The transcription factor gene was then isolated and cloned into tobacco.It was found that bHLH30 transgenic tobacco showed stronger aluminum tolerance compared with wild type tobacco.It could concluded that bHLH30 transcription factor can enhance the aluminum tolerance of plants.To clarify the mechanism of Tampa black soybean bHLH30transcription factor enhancing aluminum resistance of plants,this study selected STOP1 gene and citrate transporter gene MATE75 from Tampa black soybeans as the research object,and identified whether these were downstream genes of bHLH30transcription factor by expressed these genes into bHLH30 tobacco.Then the function of MATE75 gene that could improve aluminum resistance of plant were identified,.the main results are as follows:The STOP1 and MATE75 genes of soybean were searched on NCBI and their upstream sequences were analyzed.By finding out the distribution of cis-acting elements,the upstream sequence region which had promoter activity of the two genes were identified.The length of the two promoters were 1237 bp and 1087 bp,respectively.The DNA of Tampa black soybean seedlings treated by 50?M Al Cl₃ for 12h were used to isolated the STOP1 promoter and MATE75 promoter sequences.The plant expression vector pHGWL7-GmSTOP1P and pHGWL7-GmMATE75P were constructed by Gateway method,and transformed GmbHLH30 tobacco.Both STOP1promoter and MATE75 promoter had promoter activity in transgenic GmbHLH30tobacco.These results indicated GmbHLH30 transcription factors could regulate the expression of STOP1 and MATE75 genes.Extract RNA of Tampa black soybean seedlings which treated by AlCl₃ for 12h.Then the RNA was used to reverse transcriptional synthesis of cDNA.The plant overexpression vector pK2G7-Gm MATE75 was constructed by Gateway method,and the wild type tobacco was transformed to construct GmMATE75 transgenic tobacco line.The aluminum resistance of GmMATE75 tobacco was identified.After treating with different concentrations of Al Cl₃ for different time,the relative root elongation,citric acid content of GmMATE75 tobacco were higher than that of wild type tobacco under the same treatment condition.However the cell polysaccharide,MDA and H₂O₂ content of GmMATE75 tobacco were lower than that of the wild type tobacco.This showed that GmMATE75 tobacco has stronger resistance to aluminum than wild type tobacco,and GmMATE75 gene can improve the aluminum tolerance of plants.In summary,the GmbHLH30 transcriptional factor of black soybean can regulate the transcription factor STOP1,which further regulate the expression of the STOP1downstream gene,and regulate the promoter of the citrate channel protein MATE75gene,which promote the expression of MATE75 gene.These results improve the aluminum tolerance of the plant.