Isolation And Characterization Of TaGAPC1 Gene And The Promoter In Wheat (Triticum Aestivum L.)

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a key enzyme in glycolysis,as the cytosolic isoform of GAPDH,GAPC catalyzes the oxidation of glyceraldehyde 3-phosphate into 1,3-bisphosphoglyceric acid.However,its role against abiotic stress is largely unknown.Here we isolated the TaGAPC1 gene using the rapid amplification of c DNA ends(RACE)method,the gene structure and phylogenetic analysis of TaGAPC1 was also conducted.Through constructing the recombinant vector of TaGAPC1 and GFP gene,particle bombardment method was used in the subcellular localization of TaGAPC1 protein.Using the quantitative real-time PCR analysis,the expression patterns of TaGAPC1 gene under different stresses or in different tissues were analyzed.In addition,the TaGAPC1 promoter was isolated from wheat and it was fused to the GUS gene to construct a expression vector,the GUS staining assay was used to determine the activity of TaGAPC1 promoter after the recombinant vector was transformed into tobacco plants.The 5’deletion analysis of TaGAPC1 promoter was also carried out,to confirm the key region under certain stress,the GUS activity driven by each promoter fragment was determined under PEG8000,Na Cl,4℃ and ABA treatments.The experiment results were as followings:1.The TaGAPC1 gene was isolated from wheat by RACE method.The gene structure analysis of TaGAPC1 showed it consisted of a 1014 bp ORF,93 bp 5’UTR and 219 bp 3’UTR,12 exons and 11 introns were included in the gene.Phylogenetic analysis showed that TaGAPC1 protein shared the highest similarity with Hv GAPC1 protein from Hordem vulgare(Genbank No.P26517).2.To conduct the subcellular localization of TaGAPC1 protein,the TaGAPC1 gene was fused to GFP expression vector,then the onion epidermal cell was transformed by particle bombardment method.The result of confocal laser microscope indicated that TaGAPC1 protein localized the cytoplasm of transformed cell.3.As it is indicated by TaGAPC1 expression profiles under different stimuli,TaGAPC1 gene can be induced by PEG8000,Na Cl and ABA stresses,but the response of TaGAPC1 gene to 4 ℃was only slightly.As it is shown by TaGAPC1 expression profile in different tissues,the expression of TaGAPC1 gene in leaf is the highest,in root it is lower,while in stem it is the lowest.4.The isolated TaGAPC1 promoter was fused to GUS expression vector and then transiently transformed into tabacco plants.GUS staining analysis showed that TaGAPC1 promoter can drive the expression of GUS gene,it suggests the TaGAPC1 promoter is of activity.5.For 5’ deletion analysis of TaGAPC1 promoter,five promoter deletions were amplified and fused to the GUS expression vector,the GUS activity driven by each promoter fragments was determined under PEG8000,Na Cl,4℃ and ABA stresses after the vectors were transformed into tobacco plants.The results showed that the dehydration-responsive elements like DRE and MBS in-973 bp～-605 bp promoter region were essential for response to PEG8000 and Na Cl,the ABA-responsive element ABRE in-973 bp～-475 bp promoter region was important for response to ABA.