茶园抗性与敏感种群茶小绿叶蝉的iTRAQ蛋白质组学分析

iTRAQ proteomic analysis of insecticide resistant,and sensitive,populations of Empoasca flavescens(Hemiptera:Cicadellidae)from tea plantations

【目的】本研究旨在通过同位素标记相对和绝对定量(iTRAQ)技术,探索茶园中抗性种群(常规用药管理)与敏感种群(荒废未作管理)茶小绿叶蝉Empoasca flavescens之间的蛋白质组学差异,为后续茶小绿叶蝉的抗药性治理奠定理论基础。【方法】采用随机多点的方式采集茶小绿叶蝉,利用蛋白纯化试剂盒对总蛋白进行提取与定量。通过iTRAQ标记试剂盒对总蛋白进行酶解与标记。通过高pH-RPLC与RPLC-MS技术对多肽进行分离与鉴定。通过GO分析(Gene ontology)方法,对差异蛋白的分子功能、生物过程和细胞位置进行分析。通过KEGG Pathway网站,对差异蛋白代谢通路进行注释。通过STRING-DB数据库,对差异蛋白的互作网络进行分析。最后,对差异显著的抗性相关蛋白酶进行了分析整理。【结果】总蛋白提取结果显示,敏感与抗性种群的总蛋白含量分别为728.23μg和1261.17μg。抗性与敏感种群之间,共有1399个蛋白发生了明显变化。其中,上调与下调达到1.5倍以上的分别为101个和218个。GO分析结果显示,这319个差异蛋白具有离子结合蛋白、组成核糖体和氧化还原酶等功能,主要参与蛋白翻译、物质运输和小分子代谢过程,主要分布于细胞内膜、细胞质、核糖体和细胞核。KEGG分析结果显示,319个差异蛋白主要参与碳代谢、核糖体代谢和吞噬作用通路。STRING分析结果显示,319个差异蛋白通过多种路径相互调控。抗性相关蛋白酶整理结果显示,共有3个谷胱甘肽-S-转移酶(GSTs)、2个细胞色素P450氧化酶(P450)、2个超氧化物歧化酶(SOD)和2个过氧化物酶(POD)发生了明显变化。【结论】抗性与敏感种群茶小绿叶蝉蛋白组差异明显,差异蛋白具有多种分子功能,分布于细胞各个位置,抗性种群对药剂的解毒酶活性多数强于敏感种群。

[Objectives]To use iTRAQ(isobaric tags for relative and absolute quantification)techniques to investigate proteomic differences between resistant(conventional drug management),and sensitive(unmanaged),Empoasca flavescens populations in tea plantations,thereby providing a theoretical foundation for managing resistance in this pest.[Methods]E.flavescens were collected in tea plantations using both random and multi-point sampling methods.Proteins were extracted and quantified with a protein purification kit,then enzymatically digested and labeled with an iTRAQ labeling kit.Peptides were isolated using high pH-RPLC and identified with RPLC-MS.The molecular function,biological processes,and cell locations of differentially expressed proteins were analyzed using the GO ontology(Gene ontology)method.Metabolic pathways were annotated using the KEGG pathway website.The interaction network of differentially expressed proteins was analyzed with the String-DB database.Finally,significantly different resistance-related enzyme proteins were further analyzed and sorted.[Results]A total of 728.23μg proteins were extracted from sensitive populations and 1261.17μg from resistant populations.A total of 1399 proteins differed significantly between resistant and sensitive populations.Of these,101 were up-regulated and 218 were down-regulated more than 1.5 times.GO analysis indicates that 319 differentially expressed proteins were mainly involved in ion-binding,and in the composition of ribosomes and oxidoreductase.These proteins mainly function in protein translation,material transport and small molecule metabolic processes,and are distributed in the intracellular membrane,cytoplasm,ribosomes and nucleus,respectively.KEGG analysis indicates that the 319 differentially expressed proteins were mainly involved in the carbon and ribosomal metabolism,and the phagocytosis pathway.The results of STRING analysis indicate that the 319 differentially proteins regulated each other via multiple pathways.Analysis of resistance-related protein enzymes revealed that three glutathione S-transferase(GSTs),two cytochrome P450 oxidase(P450),two superoxide dismutase(SOD)and two peroxide dismutase(POD)were significantly different in sensitive and resistant populations.[Conclusion]Resistant and sensitive populations of E.flavescens have significant proteomic differences.Differentially expressed proteins have a variety of molecular functions and are widely distributed in different cell types.Detoxification enzyme activity was stronger in the resistant population than in the sensitive one.