Functional Analysis Of SbSTOP1 In Sweet Sorghum(Sorghum Bicolor L.)under Aluminum Stress

Soil acidity is one of the Agricultural problems all over the world.It has been estimated that 40% to 50% of potential arable soils are acidic.In China About 22.7% of the national lands are Acidic soil.Aluminum(Al)toxicity is a significant limitation to crop production worldwide in acid soils.At pH values below 5,Al3+ ions are dissolved from clay minerals and are quite toxic to plant roots,destroying the metabolism,Affecting the nutrient absorption,inhibiting root growth and function.In previous studies,different physiological and molecular mechanisms plants use to deal with Al toxicity has been identified and characterized.Two main types of Al resistance mechanisms have been documented: Al exclusion mechanisms and Al tolerance mechanisms.Many genes participate in these process,including Malate Transporters(ALMTs)?Citrate Transporters(MATEs)?ABCtransporters ?Nramp transporters,Aquaporins and transcriptional factor.AtSTOP1 and OsART1 are two related members of the C2H2-type zinc-finger transcription factor family.There are four zinc-finger domain.Both of them can positively regulate the expression of multiple Al resistance genes in Arabidopsis and rice,respectively.The study of Al resistance is relatively poor in sweet sorghum.And the homologous protein of STOP1 in Sweet sorghum has not been reported.The tolerant cultivar ROMA and the sensitive cultivar POT of sweet sorghum were used as materials.The function of SbSTOP1 was analized and the interacting proteins were detected.1.We blasted in the Sweet sorghum database with the amino acid sequence of AtSTOP1 and OsART1.There were 4 homologous genes in sweet sorghum.Sequence alignment of SbSTOP1 and orthologous proteins from other plant species was performed.The 4 zinc finger domains were conservative.All of the SbSTOP1 contain four C2H2 zinc finger domains.Phylogenic tree analysis showed that SbSTOP1 d was more closely with AtSTOP1 and ART1 than others,SbSTOP1 a,SbSTOP1b and SbSTOP1 c were relatively farther.Moreover,SbSTOP1 c was more closely with AtSTOP2.The transcriptional expression of SbSTOP1 were induced by aluminum.The transcriptional expression SbSTOP1 a,SbSTOP1b,SbSTOP1 d were induced not only by aluminum,but also cadmium.The transcriptional expression of SbSTOP1 a,SbSTOP1b,SbSTOP1 c were also induced by low pH,but SbSTOP1 d was not.2.The subcellular localization of SbSTOP1 were detected.All of the 4 SbSTOP1 localized in cell nuclei.The transcriptional activity of SbSTOP1 were examined using a yeast expression system.all of the them had transcriptional activity.3.The transcriptional regulation of SbSTOP1 d was detected in HEK293 T expression system.The results indicated SbSTOP1 d cannot regulate the expression of SbMATE,but it can regulate the expression of SbSTAR2 b and SbGlu1.The yeast library screening was performed using the N terminal of SbSTOP1 d.X-gal assay and sequencing analysis for the screening indicated that there were 16 potential proteins interacting with SbSTOP1 d.the SbSTOP1 b and SbSTOP1 d self were among the 16 proteins.Yeast Two-hybrid validation,Yeast Two-hybrid ?-galactosidase activity assay and BiFC assay showed that SbSTOP1 d can interact with SbSTOP1 b and SbSTOP1 d itself.The results indicated that SbSTOP1 can form dimers to function.4.The transgenic complementation assays was performed using the Arabidopsis Atstop1 mutant as complementation host.The results indicated that SbSTOP1 d can significantly reverse the Al3+ hypersensitivity characteristic of the Atstop1 mutant.