Expression Analysis Of Sorghum Resistance-related Gene SbSGT1 And Its Interaction With Small Molecules

Sorghum is a raw material for wine making with high economic value.However,its yield and quality are easily affected by various diseases,which limit the sustainable development of the liquor industry in Guizhou Province.Reports showed that plant disease resistance related gene SGT1 plays an important role in plant disease resistance.At present,there are few studies on SGT1 in sorghum.In this paper,the expression and molecular interaction of SbSGT1 was studied by using gene cloning,bioinformatics analysis,quantitative real-time PCR detection,prokaryotic expression,protein purification,microscale thermophoresis,transient expression and western blot.The following results were obtained in this study.1.Sorghum SbSGT1 gene cloning and bioinformatics analysis.In this study,the c DNA sequence of SbSGT1 gene was cloned.The full length of SbSGT1 sequence is 1 095 bp,encoded 364 amino acids.SbSGT1 protein is an acidic and hydrophilic protein with a molecular weight of about 40 k Da.The SbSGT1 protein contains TPR,CS and SGS conserved domains,and the protein scale solubility is 0.735.Phylogenetic analysis showed that SbSGT1 and Zm SGT1 are closely related.Promoter cis-acting elements analysis indicated that SbSGT1 has a variety of plant hormones and stress response elements.2.Sorghum SbSGT1 gene expression analysis.The q RT-PCR results showed that SbSGT1 was highly expressed in sorghum leaves,followed by stems and buds,and the lowest in roots.SbSGT1 was responding to various abiotic and biotic stresses,for example,auxin(10 ?M),salicylic acid(1 m M),abscisic acid(200 ?M),gibberellic acid(100 ?M),brassinolide(1 ?M),strigolactone(1 ?M),melatonin(200 ?M),sodium chloride(300 m M),D-mannitol(300 m M),PAMPs(flg22 and chitin),and Colletotrichum sublineola.The expression pattern of SbSGT1 is different under each of the above stresses.3.SbSGT1 protein expression,purification and interaction analysis with plant hormones.In this study,a prokaryotic expression vector p ET-28a-SbSGT1 was constructed to obtain the soluble SbSGT1.The optimal temperature for induced expression of SbSGT1 protein is 25? in E.coli BL21(DE3).At last,we got SbSGT1 protein with higher purity by using protein purification system.Our study found that SbSGT1 protein could interact with auxin(Kd=1.5934),brassinolide(Kd=3.1375),salicylic acid(Kd=3.3116),and abscisic acid(Kd=5.3839).Among them the interaction between SbSGT1 and auxin interaction was strongest.4.Analysis of the auxin on the expression of SbSGT1.In this study,the p YBA-1143-SbSGT1 plant expression vector was constructed to transiently express SbSGT1 in tobacco leaves.Then,auxin was sprayed.The results demonstrated that the expression of SbSGT1 was decreased either at the transcriptional or translational level.