中国农业科学 ›› 2019, Vol. 52 ›› Issue (7): 1237-1246.doi: 10.3864/j.issn.0578-1752.2019.07.011

• 植物保护 • 上一篇    下一篇

褐飞虱GSK-3调控糖原与海藻糖代谢的潜在功能

丁艳娟1,刘永康1,罗雨嘉1,邓颖梅1,徐红星2,唐斌1(),徐彩娣1,3   

  1. 1 杭州师范大学生命与环境科学学院,杭州 310036
    2 浙江省农业科学院植物保护与微生物研究所,杭州 310021
    3 杭州师范大学教育学院,杭州 310036
  • 收稿日期:2018-11-15 接受日期:2018-12-29 出版日期:2019-04-01 发布日期:2019-04-04
  • 通讯作者: 唐斌
  • 作者简介:丁艳娟,E-mail: dyj061004djy@163.com
  • 基金资助:
    国家自然科学基金(31672081);国家自然科学基金(31371996);国家级大学生创新创业训练计划项目(201810346031)

Potential Functions of Nilaparvata lugens GSK-3 in Regulating Glycogen and Trehalose Metabolism

DING YanJuan1,LIU YongKang1,LUO YuJia1,DENG YingMei1,XU HongXing2,TANG Bin1(),XU CaiDi1,3   

  1. 1 College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
    2 Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
    3 College of Education, Hangzhou Normal University, Hangzhou 310036
  • Received:2018-11-15 Accepted:2018-12-29 Online:2019-04-01 Published:2019-04-04
  • Contact: Bin TANG

摘要:

【目的】昆虫胰岛素信号途径能够介导糖原合成酶激酶3(glycogen synthase kinase 3,简称GSK-3或GSK3)调控体内糖原及海藻糖等糖代谢过程,从而控制昆虫的各项生命活动。论文旨在探究糖原合成酶激酶在褐飞虱(Nilaparvata lugens)体内对糖原与海藻糖代谢的调控作用。【方法】首先,基于GSK-3的cDNA编码序列,利用ExPASy工具翻译GSK-3氨基酸序列,预测蛋白分子量大小及等电点(pI);然后利用SignaIP4.1Server对其信号肽进行分析。其次,以笔者实验室饲养的褐飞虱为研究对象,从4龄开始,每12 h取材,取至成虫48 h。利用Trizol法提取褐飞虱总RNA,根据反转录试剂盒合成第一链DNA,以18S作为内参基因,通过实时荧光定量PCR(qRT-PCR)检测褐飞虱GSK-3在不同龄期mRNA水平上的相对表达量。然后利用RNAi技术,向褐飞虱体内显微注射双链RNA(dsRNA)抑制GSK-3,以注射dsGFP的褐飞虱作为对照组。注射后48 h利用qRT-PCR技术检测GSK-3的表达情况,确定抑制效果。另外,取注射后48 h虫体,分别测定褐飞虱体内海藻糖、葡萄糖、糖原含量及海藻糖酶(trehalase,TRE)活性变化。最后采用qRT-PCR检测胰岛素信号通路胰岛素受体基因(insulin receptor,InR)、类胰岛素多肽基因(insulin-like peptides,Ilps)及海藻糖代谢途径TRE、海藻糖合成酶基因(trehalose-6-phophate synthase,TPS)、糖原磷酸化酶基因(glycogen phosphorylase,GP)、糖原合成酶基因(glycogen synthase,GS)中相关基因的表达,分析GSK-3在胰岛素信号通路及海藻糖代谢途径中的调控作用。【结果】褐飞虱GSK-3开放阅读框为1 914 bp,编码637个氨基酸;预测蛋白分子量为69.25 kD,等电点为9.15,为偏碱性蛋白,无信号肽结构,序列高度保守。发育表达模式结果显示GSK-3在不同发育阶段表达不一致,5龄若虫蜕皮前后低表达。GSK-3的dsRNA注射后48 h,与对照组dsGFP相比,GSK-3表达极显著下降,表明RNA干扰效果明显。糖原含量和两类海藻糖酶活性显著下降,而海藻糖含量显著上升,推测糖原和葡萄糖转化为海藻糖,作为其生理活动的能量来源。qRT-PCR检测发现,当GSK-3表达抑制后48 h,TRE1-2的表达量显著下降,而TRE1-1TRE2的表达量极显著下降。另外,2个TPS基因、GS以及GP的表达量均极显著下降;胰岛素信号通路的2个InR基因和4个Ilps基因的表达同样被抑制,间接表明InR能够调控GSK-3的表达。【结论】褐飞虱GSK-3低表达后能够通过调控胰岛素信号通路及海藻糖代谢途径相关基因表达来调控糖原及海藻糖代谢。相关研究结果有助于更加全面地探索褐飞虱等昆虫糖原合成酶激酶调控海藻糖及糖类物质平衡的潜在分子机理。

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)