Trypsin can significantly improve the storage quality of Hylocereus undatus (H. undatus). To verify the hub WRKY gene of H. undatus in trypsin preservation, joint analysis of transcriptome and protein–protein interaction (PPI) network was carried out, and virus‐induced gene silencing (VIGS) was conducted. In the transcriptome of H. undatus, GO directed acyclic graph (DAG) showed that the GO terms of 55 WRKY genes were mainly enriched in sequence‐specific DNA binding, DNA binding transcription factor activity, and so on. The GO enrichment chord diagram showed that HuWRKY40 was significantly up‐regulated in the enriched top10 GO terms. KEGG enrichment analysis showed that 55 WRKY genes were mainly enriched in plant–pathogen interaction and MAPK pathway. The results of PPI network showed that HuWRKY40 was a hub protein of WRKY transcription factors (TFs) family regulated by trypsin, which was consistent with the results of transcriptome analysis. Bioinformatics analysis showed that HuWRKY40 of H. undatus had the highest homology with Beta vulgaris L. and Spinacia oleracea L. The function of the core regulatory protein HuWRKY40 was further clarified by VIGS technology. The results of VIGS showed that there was a big difference between the phenotype of the pTRV2‐HuWRKY40 group and that of the control group. Finally, it was confirmed that HuWRKY40 accelerated the synthesis of flavonoids and improved the fruit quality during the storage of H. undatus. This study found that trypsin may regulate HuWRKY40 activity through the MAPK cascade pathway, affect the participation of flavonoid synthesis, and then delay fruit corruption. Practical applications With attention of people to the safety and freshness of fruits and vegetables, biological preservation technology has become one of the hotspots in the field of preservation in recent years. Trypsin can significantly improve the antioxidant capacity of fruits and vegetables. As a new biological preservative, it is convenient to operate and economical. In the current work, the mechanism of trypsin on the WRKY TFs during H. undatus storage was investigated. The application of trypsin would provide a new strategy for the storage quality control of fruits and vegetables.

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