Functional Analysis Of TaGAPA Gene And Promoter In Chinese Spring Wheat

The chloroplastic Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a tetramer encoded by GAPA gene in Chinese Spring wheat(Triticum aestivum L.).Different from the role of cytoplasmic GAPDH in glycolysis,chloroplastic GAPDH participates in photosynthetic CO2 fixation by reducing 1,3-Bisphosphoglyceric acid to glyceraldehyde-3-phosphate.Recently,it was found that cytoplasmic GAPDH was involved in stress response in plants.Previous researches indicated that TaGAPA gene expression was notable change under abiotic stresses.The notion of this paper was to explore the stress-resistant function of GAPA by analysis TaGAPA genes and their promoters function.In order to explore the biological function of these genes,TaGAPA genes and their promoter sequences were cloned and identified in Chinese Spring Wheat.For analyzing gene functions,we identified GAPA gene subcellular localization by using Laser confocal microscopy and detected expression level of GAPA gene under stresses by q RT-PCR.For identifying the promoter functions,we predicted cis-elements of GAPA promoter by Plant CARE software and analyzed the promoter activity by constructing transient expression vectors.This paper provides foundation to explore GAPA gene functions to response stresses.The results are as follows,1.Three GAPA genes located at A,B or D subgenome were found in Chinese Spring Wheat.The identity of three GAPA gene amino acid is up to 97.42%,therefore only one CDS sequence was cloned for this research.This CDS sequence is1206 bp,and encodes 402 amino acids.Three TaGAPA promoter was cloned,and the length of three promoter was 1677 bp,1917bp or 2040 bp respectively.They were named TaGAPA1 promoter,TaGAPA2 promoter and TaGAPA3 promoter respectively.The identity of three GAPA promoters we cloned was 50.98%.The prediction of cis-elements showed that there are many cis-elements located in three promoter sequences and the quantity of light responsive element is the most.However,the species and quantity of cis-elements located in each promoter is notably different.In detail,TaGAPA1 promoter’s light responsive elements is the most,both TaGAPA2 promoter and TaGAPA3 promoter have many drought and low temperature responsive elements.Especially,TaGAPA2 promoter has more ABA responsive elements than TaGAPA2 promoter and TaGAPA3 promoter.2.Subcellular localization vector pCMBIA1302-TaGAPA-GFP was constructed and transfected to tobacco protoplast by transient transfection,and then observated the fluorescent under Laser confocal microscopy.The result indicated that GAPA protein located in chloroplast,and GAPA is a chloroplast protein.3.The temporal expression patterns of TaGAPA gene was detected by qRT-PCR under PEG,H2O2,ABA or 4 ℃ treatment at different treat time.The result indicated that TaGAPA gene could response all treatments,and the best response was H2O2 stress,and ABA stress took the second place.This assay declared that TaGAPA gene can respond stresses.4.Three promoters were constrcucted transient expression vector with GUS lable,and then applied to histochemical staining assay and GUS activity assay under different stresses.The results elicudated three promoters had promoter activity,and displayed different responsive ability under different stresses.In GUS activity assay,the sharpest response of ABA stress was TaGAPA2 promoter.5.Three promoters were constrcucted transient expression vector with Dual-Luciferase vector.TaGAPA promoter activity and ability of response stress were estimated by testing radio of Fluc and Rluc.The results showed that TaGAPA1 and TaGAPA2 promoter could response ABA and 4℃stresses,TaGAPA3 promoter could response 4℃stress but ABA stress.