关键词: 捕蝇草, 橘小实蝇, 生物农药, 丝氨酸羧肽酶, 杀虫活性

Abstract:

【Background】The long-term use of chemical pesticides has led to a series of problems such as drug resistance and environmental residue. Therefore, the green control of pest requires safer and more efficient active substances. Plants are the largest source of active substances of natural products for pest control. Carnivorous plants have the ability to capture and digest insects, and the components of their secreted digestive fluids exhibit potential insecticidal activity. 【Objective】The purpose of this study is to identify the protein components in the digestive fluid of Dionaea muscipula, and to screen out the proteins with insecticidal activity by combining the transcriptome data of D. muscipula after being induced by prey. 【Method】The adults of Bactrocera dorsalis were put into the trap to observe and record the process of digestion, and confirm the digestive ability of the trap. By analyzing the transcriptome data of the traps and glands of D. muscipula, genes with stress expression in response to prey stimulation were identified. Combined with the proteomic sequencing of the digestive fluid within the traps, the proteases that play a crucial role in the digestion process were clarified. Recombinant proteins were expressed in Escherichia coli in vitro, and injected into or dripped onto B. dorsalis adult to test insecticidal activity of recombinant proteins. 【Result】By observing the process of D. muscipula digesting B. dorsalis, it was found that the D. muscipula has the ability to digest B. dorsalis. The secreted digestive fluid can damage the body wall of B. dorsalis, and it takes about 10 to 13 days to digest a B. dorsalis. Following enzymatic digestion, only the epidermal layer is left. After being stimulated by the prey, DNA-binding transcription factors, serine carboxypeptidase, calmodulin and K⁺ uptake protein were simultaneously identified in the differentially expressed genes of the transcriptome and proteome datasets, and they may play an important role in the process of digesting prey by D. muscipula. The expression levels of serine carboxypeptidase genes DmSCBP1, DmSCBP2, DmSCBP5, DmSCBP8 in the traps and glands significantly increased after being stimulated by the prey. Among them, the expression level of DmSCBP1 was the most significantly increased, and the protein sequence of this gene was also detected in the protein sequencing of the digestive fluid. The recombinant protein of DmSCBP1 was expressed through the prokaryotic expression system. The biological assay results showed that this protein had no contact toxicity on B. dorsalis, but the injection method could cause a high mortality, and the mortality increased with the increase of time and dose within a certain range. 【Conclusion】The recombinant DmSCBP1 has lethal activity against the B. dorsalis, but its effective usage methods need further improvement.

Key words: Dionaea muscipula, Bactrocera dorsalis, biopesticide, serine carboxypeptidase, insecticidal activity