Backgrounds: Wheat (Triticum aestivum L.) is one of the most important food crops in the world. It faces various abiotic stresses during its growth. Drought is one of the main factors limiting the growth and development of wheat. Severe drought stress will Lead to a decline in wheat production. Cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is an important member of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) family, which is widely present in plant cytoplasm. Plants play an important role in the process of primary metabolism and stress resistance.Result: In this study, a comparative transcriptomic analysis of the TaGAPCs-RNAi strain of Changwu 134 and the wild-type wheat seedlings of Changwu 134 under natural drought conditions was carried out. A total of 30067 differentially expressed genes were screened in RNAi strains and wild-type strains, of which 19,959 genes were up-regulated in RNAi strains and 10,108 genes were down-regulated in transcription. GO analysis shows that differential genes are mainly enriched in biological regulation, cellular processes, metabolic processes, and responses to stimuli. KEGG analysis showed that the differential genes were mainly concentrated in the biosynthesis of phenylpropane, plant hormone signal transduction and flavonoid biosynthesis pathways. By analyzing the expression levels of differential transcription factors, the significantly down-regulated transcription factor WRKY family member TaWRKY2 / 22/28/29/33/40/47/52 in wheat was screened out. The TaWRKY28/33/40/47 gene silencing line was successfully obtained using the barley stripe mosaic virus (BSMV-VIGS) technology. The plants with TaWRKY28/33/40/47 gene silenced were subjected to natural drought treatment, and physiological and biochemical index tests were carried out. The results showed that the growth status of gene-silenced plants was worse than that of wild-type plants, and the relative water content and chlorophyll content decreased. The content of MDA, H2O2 and superoxide anion increases, the activity of antioxidant enzymes (SOD, POD, CAT) decreases, and the content of proline decreases. Conclusion: The results showed that TaGAPCs regulates the expression of some TaWRKYs transcription factors, activates antioxidant pathways, enhances tolerance of wheat to drought stress.