关键词: 褐飞虱, RNA干扰, 糖原合成酶激酶3, 糖原与海藻糖代谢, 实时荧光定量PCR（qRT-PCR）

Abstract:

【Objective】The insect insulin signaling pathway can mediate glycogen synthase kinase 3 (GSK-3 or GSK3) to regulate glucose metabolism in the body, such as glycogen and trehalose, thereby controlling different life activities of insects. The objective of this study is to explore the regulation of glycogen synthase kinase on the metabolism of glycogen and trehalose in Nilaparvata lugens.【Method】 Firstly, based on the cDNA coding sequence of GSK-3, the GSK-3 amino acid sequence was translated using the ExPASy tool to predict the molecular weight and isoelectric point (pI) of the protein, and then the signal peptide was analyzed by SignalaIP4.1Server. Secondly, the N. lugens raised in the author’s laboratory was collected every 12 hours from the 4th instar to 48-h-old adult. The total RNA of N. lugens was extracted by Trizol method. The first strand DNA was synthesized according to the reverse transcription kit, and 18S was used as the internal reference gene. The relative expression of GSK-3 in different ages at mRNA level was detected by quantitative real-time PCR (qRT-PCR). Then, double-stranded RNA (dsRNA) was microinjected into N. lugens with RNAi technology to inhibit the GSK-3, and N. lugens of dsGFP was injected as a control group. The expression of GSK-3 was detected by qRT-PCR 48 h after injection to determine the inhibitory effect. In addition, the N. lugens was taken 48 h after injection, and the change of trehalose, glucose, glycogen content and trehalase (TRE) activity in N. lugens was determined. Finally, the relative expression of related genes in insulin signaling pathway (including insulin receptor (InR), insulin-like peptides (Ilps)) and trehalose metabolism pathway (TRE, trehalose-6-phophate synthase (TPS), glycogen phosphorylase (GP), glycogen synthase (GS)) was detected by qRT-PCR to analyze the regulation of GSK-3 in insulin signaling pathway and trehalose metabolic pathway.【Result】The open reading frame of N. lugens GSK-3 is 1 914 bp, encoding 637 amino acids; the predicted molecular weight of the protein is 69.25 kD, and the isoelectric point is 9.15. It is a basic protein with no signal peptide structure and the sequence is highly conserved. The results of developmental expression pattern showed that the expression of GSK-3 was inconsistent at different developmental stages, and the expression was low before and after molting of 5th instar nymph. At 48 h after the dsRNA injection of GSK-3, the expression of GSK-3 decreased significantly compared with the dsGFP of the control group, indicating that the RNA interference effect was obvious. Glycogen content and two types of trehalase activity decreased significantly, while trehalose content increased significantly. It is speculated that glycogen and glucose are converted to trehalose as an energy source for their physiological activities. The results of qRT-PCR showed that the expression of TRE1-2 significantly decreased 48 h after the inhibition of GSK-3 expression, while the expression of TRE1-1 and TRE2 extremely significant decreased. In addition, the expression of two TPS genes, GS and GP genes all extremely significant decreased; the expression of two InR genes and four Ilps genes in the insulin signaling pathway were also inhibited, indirectly indicating that InR can regulate the expression of GSK-3. 【Conclusion】 The low expression of GSK-3 in N. lugens can regulate glycogen and trehalose metabolism by regulating insulin signaling pathway and trehalose metabolism pathway related gene expression. The relevant research results will help to explore more comprehensive molecular mechanisms for the regulation of the balance of trehalose and carbohydrates by insect glycogen synthase kinases such as N. lugens.

Key words: Nilaparvata lugens, RNA interference (RNAi), glycogen synthase kinase 3, glycogen and trehalose metabolism, quantitative real-time PCR (qRT-PCR)