Promoter Cloning And Functional Verification Of Salt-induced And Root-preferential Expressed Gene In Wheat

Wheat is one of majorcereal cropsfor mankind,and salt stress can significantly reduce wheat yield and quality.There are many difficulties in using conventional breeding methods to improve the salt tolerance of wheat in molecular breeding research.It is necessary to identify the salt-tolerant genes in wheatfor molecular breeding.In this study,bioinformatics method was used to select two salt response candidate genes from 425 gene probes.The wheat variety Chinese spring was used as experimental material for salt stress treatment,and the expression pattern of candidate genes was verified by PCR.The gene corresponding to probe Ta.5463.1.A1＿at was proved preferentially expressed in roots and significantly up-regulated after salt stress induction.URGIBlastfound tahtthe gene located at wheat B genomeshared the highest homology with the probeand was designated as Tasip.Promoters Tasipro3 and promoter Tasipro5 were cloned using the specific primersdesigned byonline primer designingsoftware.The results show that the bioinformatics method can effectively screen genes that can respondto specific stress conditions in plant specific tissues,and improve the accuracy of gene screening.To further verify the function of the promoters Tasipro3 and Tasipro5,the present study constructed an expression vector p UKGS-Bar withuseful features.The vector has Gateway cloning elements for promoterrecombinaion,Ubiquitin promoter for gene expression in cereals,Bar selective marker gene for positive plant screening,and the GUS reporter marker gene is driven by the candidatepromoter,so that it can be verified by GUS histochemical staining methodand subsequentfunctional analysis.The cloned promoters Tasipro3,Tasipro5 wer ligated with expression vector p UKGS-Bar by Gateway cloning,respectively.Arabidopsis plants were transformed by Agrobacterium-mediated floral dip transformation.The results of GUS staining showed that the promoters Tasipro3 and Tasipro5 were induced by salt and expressed preferntially at the root.These promotersdemonstrated great application potential in the salt-tolerant molecular breeding of wheat.