Molecular Mechanism Of Tamba Black Soybean GmbHLH30 And Arabidopsis AtPrx64 Gene Enhancing Plant Tolerance To Aluminum

Aluminum is the most abundant metal elements in the earth.It will be released in the form of Al3+ from the acid soil with pH<5,which has a toxic effect on plants and affects the growth of crops.With the degree of soil acidification rises all over the word,more than 50%of the arable soil is acidic,so aluminum toxicity has become a major factor limitating the yield of crops.The research on aluminum tolerance in plants has become the focus on agricultural production.Our previous study found that GmbHLH30 was up-regulated in the SSH cDNA Library of Tamba black soybean treated with aluminum.Then,the GmbHLH30 gene was identified from the roots of Tamba black soybean and it was also found that overexpression of GmbHLH30 in tobacco could increase the resistance of tobacco to aluminum.The homozygous of T-DNA insertion mutants was screened out by primers designing and GmbHLH30 mutation was detected on the levels of RNA and DNA.Meanwhile,transgenic Arabidopsis,in which GmbHLH30 was overexpressed by transfected with pK2G7-GmbHLH30 vector was screened.The up-regulated and down-regulated genes were clustered through DNA chip hybridization technology,in which 12 genes of interest were selected and the E-box and G-box domains in the promoter of these genes were analyzed.In this study,the molecular mechanisms of GmbHLH30 in improving the resistance to aluminum stress in black soybean was analyzed.At the same time,the effect of overexpression of Arabidopsis AtPrx64 on the aluminum tolerance in trangeic tobacco was also analyzed,and its mechanism was discussed.Main results were as follows:The difference of the expression of GmbHLH30 on transcriptional level and protein level between the roots and leaves of Tamba black soybean was analyzed by fluorescence quantitative PCR and Western blotting.The results showed that the expression of GmbHLH30 in the root of Tamba black soybean was induced under aluminum stress,and with the increasing concentration of Al,its expression went up significantly.Whereas the expression of GmbHLH30 in leaves was very low and could only be induced slightly by aluminum.The results suggested that the expression of GmbHLH30 in roots could be induced by aluminum,which has tissue specificity.The subcellular localization of the GmbHLH30 was also examined by expressing GmbHLH30-GFP fusion protein transiently in onion epidermal cells.It was showed that GmbHLH30-GFP expressed in nucleus was observed by the confocal laser microscopy,indicating that GmbHLH30 was localized in the nucleus.The target genes which may be regulated by GmbHLH30 were identified by chromatin immunoprecipitation.Three genes,GmMATE,GmSTOP1 and GmALMT1,were identified in the related genes corresponding to aluminum stress of the interest.Two promoter vectors pHGWL7-pGmALMT1-LUC and pHGWL7-pGmMATE-LUC were constructed respectively.Then the transient expression of pHGWL7-pGmMATE-LUC and pHGWL7-pGmALMT1-LUC in the leaves of transgenic GmbHLH30 tobacco was detected,the activity of LUC could be detected in the transient expression of pHGWL7-pGmMA7E-LUC,but the activity of LUC in the transient expression of pHGWL7-pGmALMT1-LUC was much lower than in pHGWL7-pGmMATE-LUC.The results suggest that GmbHLH30 mainly has transcriptional activity on the promoter of GmMATE,which regulates the transcription of GmMATE by binding to the promoter of GmMATE.GmMATE in the Tamba black soybeans was isolated and identified according to the sequence of Glycine max aluminum-activated citrate transporter?named GmMATE,mRNA,NM₀01251793?.It was found that the gene has deletion and mutation in Tamba black soybean,so it was named GmMATE-TBl;Compared with the sequence of GmMATE?NM₀01251793?,the bases of No.525 to 530 was deleted,No.30 base was mutated from T to A,No.204,232,1331,1409 changed from C to T,and No.1538 base from T to C.These deletions and mutations also resulted in the changes in protein sequence it encodes.Results of model-building ananlysis on the structure of GmMATE and GmMATE-TB1 showed that the change of the protein sequence of GmMATE-TB1 had a great influence on the extracellular structure,but the effect on its function was unknown.A plant expression vector was also constructed,some research on the subcellular localization of GmMATE-TB1 protein had been done and its possible functions was also be analyzed.The expression levels of GmbHLH30 and GmMATE-TBl genes under aluminum,cadmium and manganese stress in the roots of Tamba black soybean were compared.It was found that under aluminum and cadmium stress,these two genes were up-regulated significantly,while under manganese stress conditions,the increase rates of the expression of GmbHLH30 and GmMATE-TB1 were lower,and the increase rate of GmMATE-TB1 is more obvious than that of GmbHLH30.The results suggested that the way of plant on metal tolerance controlled by GmMATE-TBl might be a common pathway under the stress of these metals.Meanwhile,it also suggested that there might be other mechanisms to regulate the the expression of GmMATE-TBl besides the regulation of GmbHLH30 on the expression of GmMATE-TBl.The mechanism of soybean Tamba black response to different metal stress conditions might be different which could be possibly caused by the different effects of these metals on plants.In addition,the function of Arabidopsis AtPrx64 gene to A1 stress had been studied in this paper.Previous studies in our laboratory showed that overexpression of AtPrx64 gene increased the root growth and reduced the accumulation of ROS in the root tips.Compared with wild type tobaccos,transgenic tobaccos had much less malondialdehyde and H2O2 in the roots,much more soluble proteins and root citrate exudation.The activity of plasma membrane?PM?H+-ATPase,the phosphorylation of PM H+-ATPase and its interaction with 14-3-3 proteins increased in transgenic tobaccos,which might improve the citrate exudation;In this study,the results showed that compared with wild type tobaccos,the accumulation of Al in root tips of transgenic tobaccos decreased,while the content of lignin increased.Taken together,these results showed that overexpression of AtPrx64 gene might enhance the tolerance of tobacco to A1 stress through decreasing the accumulation of A1 by increasing the citrate exudation and influencing the structure of the cell wall.