Abstract: 【Objective】Pyruvate kinase (PYK) is a glycolytic enzyme that regulates the balance between glycolysis and gluconeogenesis, so it has an important physiological role in insects. This study aims to explore the biological functions of HaPYK in Harmonia axyridis by analyzing the sequence structure of the three HaPYKs and RNA interference technology. 【Method】Based on the three HaPYK sequences obtained from the whole genome of H. axyridis (named HaPYK1-1, HaPYK1-2, HaPYK2, respectively), we analyzed its physicochemical properties, sequence structure and homology with other insects through bioinformatics knowledge. Then, the dsRNA synthesized in vitro was injected into the adult on the first day of emergence by microinjection. The experimental materials were collected 48 and 72 h after injection, and the total RNA extraction and reverse transcription experiments were performed. In order to evaluate the inhibitory effect, we used real-time fluorescent quantitative PCR technology to determine the expression levels of three HaPYKs. Finally, the expression levels of genes related to trehalose metabolism, the content of glucose, glycogen, trehalose, and the activities of two trehalase enzymes were detected. 【Result】The experimental data showed that the open reading frames of HaPYK1-1, HaPYK1-2 and HaPYK2 were 1 350, 1 569 and 1 656 bp, respectively. The protein sizes were 449, 522 and 551 aa, and the theoretical isoelectric points were 5.04, 5.02 and 5.01, respectively. The conserved domains predicted that the three HaPYKs belonged to the pyruvate_kinase superfamily. The secondary structure of HaPYK was mainly α-helix and random coil, but also contained extended chain and β-corner. The oligomer types of the three sequences were all homologous tetramer. The phylogenetic tree showed that HaPYK was closely related to the same coleoptera insects, such as Leptinotarsa decemlineata and Tribolium castaneum. In addition, after injection of dsHaPYK1-1, the expression levels of HaPYK1-2 and HaTRE1-4 were significantly down-regulated, while the expression levels of HaPYK2, HaTRE1-1, HaTRE1-2, HaTRE2-like, and HaTPS were significantly up-regulated; after injection of dsHaPYK1-2, the expression of HaTRE1-1, HaTRE1-2, HaTRE1-3, HaTRE1-5, HaTRE2, HaTPS increased significantly; after injection of dsHaPYK2, only HaTRE1-1 was significantly up-regulated at 48 h, and the expression levels of HaTRE1-3, HaTRE1-4, HaTRE1-5, HaTRE2, HaTRE2-like were down-regulated. Compared with the control group, in the dsHaPYK1-2 group, the activity of TRE1 reduced significantly at 48 h, and the content of trehalose and glycogen was increased significantly; in dsHaPYK1-1 or dsHaPYK2, the glucose content decreased at 48 h, the glucose and trehalose content increased at 72 h, and the enzyme activity of TRE2 was increased significantly; at 48 h in dsHaPYK2 group, trehalose significantly reduced, but a significant increase in glycogen content.【Conclusion】There are three HaPYKs in H. axyridis. By injecting exogenous dsHaPYK, the expression level of target genes can be successfully reduced. Suppressing their expression levels could affect the metabolism of trehalose, and the regulatory mechanisms of the three HaPYKs are not the same. The above results provide theoretical basis and help for further research on the energy metabolism of H. axyridis and other biological functions of HaPYK in H. axyridis.

Key words: Harmonia axyridis; bioinformatics; pyruvate kinase (PYK), trehalose metabolism; RNA interference (RNAi)