抗氟苯虫酰胺小菜蛾差异表达基因及其通路

doi:10.3864/j.issn.0578-1752.2018.11.008

摘要/Abstract

摘要： 【目的】随着氟苯虫酰胺等双酰胺类杀虫剂的广泛应用，小菜蛾（Plutella xylostella）对该类药剂的抗性愈发突出。研究旨在从转录组水平研究小菜蛾对氟苯虫酰胺的抗性机制，明确小菜蛾对氟苯虫酰胺产生抗性的关键基因及通路，为揭示小菜蛾对氟苯虫酰胺的抗性机制打下基础。【方法】以室内筛选的小菜蛾抗氟苯虫酰胺品系（Rh28）、田间抗性种群（Rz36）和敏感品系（S）为材料，通过转录组测序技术（RNA-Seq），获得抗性小菜蛾品系/种群在转录水平上的差异表达基因及抗性显著上调表达基因，采用基因本体（GO）显著性富集分析及京都基因与基因组百科全书数据库（KEGG）富集分析，确定差异表达基因具有的主要生物学功能及其参与的主要生化代谢和信号转导途径。【结果】通过将不同抗性品系/种群与敏感品系的RNA-Seq数据对比，获得数量不等的差异表达基因。将差异基因富集的生物学过程（biological process）和信号通路（pathway）进行GO分析，发现其主要集中于对刺激物的反应（response to stimulus）、催化活性（catalytic activity）和代谢过程（metabolic process）等条目中；而KEGG分析发现，差异基因主要集中于代谢通路（metabolic pathway）中，基因数量占比最高，为17.84%。通过统计分析进一步获得了抗性显著上调表达基因，并对其中218个基因开展了GO分析，其仍然主要集中在代谢过程、对刺激物的反应和生物调节（biological regulation）等条目中；通过对抗性显著上调基因进行KEGG分析，发现其仍主要集中在代谢通路中，其中谷胱甘肽-S-转移酶、细胞色素P450解毒酶及参与昆虫多种生理反应的热激蛋白（Hsp）超基因家族等基因的表达量均显著上调。通过聚类热图分析发现，显著表达上调的基因较多集中在内质网上蛋白质的加工（protein processing in endoplasmic reticulum）、酪氨酸代谢（tyrosine metabolism）、咖啡因代谢（caffeine metabolism）和Wnt信号通路（Wnt signaling pathway）中。【结论】获得了小菜蛾抗氟苯虫酰胺差异表达基因及抗性显著上调表达基因，其主要富集于代谢过程、应激反应及对刺激物的反应等条目中，这些基因的相互协同调控是小菜蛾对氟苯虫酰胺产生抗性的重要机制。

关键词: 转录组测序, 差异表达基因, 氟苯虫酰胺, 抗性, 小菜蛾

Abstract: 【Objective】With the widespread use of diamide insecticides, such as flubendiamide, the resistance of Plutella xylostella to these insecticides became more and more prominent. The objective of this study is to research the resistant mechanism to flubendiamide in P. xylostella from the transcriptome perspective, identify the key genes and pathways that lead to resistance to flubendiamide in P. xylostella, and to lay a foundation for revealing the mechanism of resistance to flubendiamide in P. xylostella.【Method】 The resistance to flubendiamide in P. xylostella was determined by the RNA sequencing technique (RNA-Seq) using the flubendiamide-resistant strains (R_h28), the field resistant population (R_z36) and the susceptible strain (S). The differential expressed genes (DEGs) and significantly up-regulated resistant genes were obtained in the strains/populations. The enrichment analysis of Gene Ontology (GO) and the enrichment analysis of Kyoto Encyclopedia of Gene and Genome (KEGG) were used to identify DEGs. The major biological functions, the major biochemical metabolic and signaling pathways of the DEGs were elaborated. 【Result】The different numbers of DEGs were obtained by comparing RNA-Seq data of the different resistant strains/populations with susceptible strains. GO analysis of the biological process (BP) and pathway for differential gene enrichment showed that they were mainly focused on response to stimulus, catalytic activity and metabolic process items. KEGG analysis found that the differential genes were mainly concentrated in the metabolic pathway, with the highest number of genes (17.84%). the genes with significantly up-regulated expression were further obtained, and GO analysis was carried out on 218 of them, which still mainly focused on the metabolic process, response to stimulus and biological regulation items. KEGG analysis of significantly up-regulated expression genes found that the DEGs were still mainly in the metabolic pathway. The genes of glutathione-S-transferase, cytochrome P450 detoxification enzymes, and involved in a variety of insect physiological responses to heat shock protein (Hsp) family genes were significantly up-regulated compared with the susceptible strain. The results also showed that most of the genes with significant up-regulation were concentrated in protein processing in endoplasmic reticulum, tyrosine metabolism, caffeine metabolism and the Wnt signaling pathway by heat map and clustering analysis.【Conclusion】the DEGs and the significantly up-regulated expression genes were obtained in resistant strain of P. xylostella, which of them were mainly involved in the metabolic process, the response to stress and the response to stimulus. The synergistic regulation of these genes is an important mechanism of resistance to flubendiamide in P. xylostella.

Key words: RNA sequencing, differential expressed genes, flubendiamide, resistance, Plutella xylostella

王成花，孙诗晴，徐巨龙，赵小龙，薛超彬. 抗氟苯虫酰胺小菜蛾差异表达基因及其通路[J]. 中国农业科学, 2018, 51(11): 2106-2115.

WANG ChengHua, SUN ShiQing, XU JuLong, ZHAO XiaoLong, XUE ChaoBin. Differential Expressed Genes and their Pathways of the Resistance to Flubendiamide in Plutella xylostella[J]. Scientia Agricultura Sinica, 2018, 51(11): 2106-2115.

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参考文献

[1] SUKONTHABHIROM S, DUMRONGSAK D, JUMROON S, SAROCH T, CHAWENG A, TANAKA T. Update on DBM diamide resistance from the Thailand: situation and causal factors// Srinivasan R, Shelton A M, Collins H L. The sixth international workshop on management of the diamondback moth and other crucifer insect pests. AVRDC-The World Vegetable Center, 2011: 202-206.

[2] TROCZKA B, ZIMMER C T, ELIAS J, SCHORN C, BASS C, DAVIES T G E, FIELD L M, WILLIAMSON M S, SLATER R, NAUEN R. Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor. Insect Biochemistry and Molecular Biology, 2012, 42(11): 873-880.

[3] STEINBACH D, GUTBROD O, LÜMMEN P, MATTHIESEN S, SCHORN C, NAUEN R. Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella. Insect Biochemistry and Molecular Biology, 2015, 63: 14-22.

[4] YAN H H, XUE C B, LI G Y, ZHAO X L, CHE X Z, WANG L L. Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.). Pesticide Biochemistry and Physiology, 2014, 115: 73-77.

[5] UCHIYAMA T, Ozawa A. Rapid development of resistance to diamide insecticides in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae), in the tea fields of Shizuoka Prefecture, Japan. Applied Entomology and Zoology, 2014, 49(4): 529-534.

[6] RODITAKIS E, VASAKIS E, GRISPOU M, STAVRAKAKI M, NAUEN R, GRAVOUIL M, BASSI A. First report of Tuta absoluta resistance to diamide insecticides. Journal of Pest Science, 2015, 88(1): 9-16.

[7] TROCZKA B J, WILLIAMS A J, WILLIAMSON M S, FIELD L M, LÜMMEN P, DAVIES T G E. Stable expression and functional characterization of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides. Scientific Reports, 2015, 5: 14680.

[8] WANG X L, WU Y D. High levels of resistance to chlorantraniliprole evolved in field populations of Plutella xylostella. Journal of Economic Entomology, 2012, 105(3): 1019-1023.

[9] HU Z D, FENG X, LIN Q S, CHEN H Y, Li Z Y, YIN F, LIANG P, GAO X W. Biochemical mechanism of chlorantraniliprole resistance in the diamondback moth, Plutella xylostella Linnaeus. Journal of Integrative Agriculture, 2014, 13(11): 2452-2459.

[10] GUO L, LIANG P, ZHOU X G, GAO X W. Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.). Scientific Reports, 2014, 4: 6924.

[11] SUN L N, CUI L, RUI C H, YAN X J, YANG D B, YUAN H Z. Modulation of the expression of ryanodine receptor mRNA from Plutella xylostella as a result of diamide insecticide application. Gene, 2012, 511: 265-273.

[12] LI X X, GUO L, ZHOU X G, GAO X W, LIANG P. miRNAs regulated overexpression of ryanodine receptor is involved in chlorantraniliprole resistance in Plutella xylostella (L.). Scientific Reports, 2015, 5: 14095.

[13] LI B, DEWEY C N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics 2011, 12(1): 323. ,

[14] AUDIC S, CLAVERIE J M. The significance of digital gene expression profiles. Genome Research, 1997, 7(10): 986-995.

[15] BENJAMINI Y, YEKUTIELI D. The control of the false discovery rate in multiple testing under dependency. The Annals of Statistics, 2001, 29(4): 1165-1188.

[16] Ye J, Fang L, Zheng H K, Zhang Y, Chen J, Zhang Z J, Wang J, LI S T, LI R Q, Bolund L, Wang J. WEGO: a web tool for plotting GO annotations. Nucleic Acids Research, 2006, 34(2): W293-W297.

[17] 马晶晶, 章涛. PPARγ功能与疾病关系研究进展. 中国药理学通报, 2012, 28(5): 601-604.

MA J J, ZHANG T. Function of PPARγ and its relationship with diseases: a present review. Chinese Pharmacological Bulletin, 2012, 28(5): 601-604. (in Chinese)

[18] 郭直岳, 王国红, 甄静, 贺秉军. 氯虫酰胺对甜菜夜蛾神经元钠离子通道电流的影响. 河南农业科学, 2013, 42(10): 79-83.

GUO Z Y, WANG G H, ZHEN J, HE B J. Effects of chlorantraniliprole on sodium channel currents of central neurons from Laphygma exigue. Journal of Henan Agricultural Sciences, 2013, 42(10): 79-83. (in Chinese)

[19] HAYES J D, FLANAGAN J U, JOWSEY I R. Glutathione transferases. Annual Review of Pharmacology and Toxicology, 2005, 45: 51-88.

[20] FEYEREISEN R. Insect P450 enzymes. Annual Reviews of Entomology, 1999, 44: 507-533.

[21] ZHANG L, GAO X, LIANG P. Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae).Pesticide Biochemistry and Physiology, 2007, 89: 65-72.

[22] GEHRMANN M, BRUNNER M, PFISTER K, REICHLE A, KREMMER E, MULTHOFF G. Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs. Clinical Cancer Research, 2004, 10: 3354-3364.

[23] KIM K K, KIM R, KIM S H. Crystal structure of a small heat-shock protein. Nature, 1998, 394: 595-599.

[24] FRANCK E, MADSEN O, VAN RHEEDE T, RICARD G, HUYNEN M A, DE JONG W W. Evolutionary diversity of vertebrate small heat shock proteins. Journal of Molecular Evolution, 2004, 59: 792-805.

[25] QIN W, NEAL S J, ROBERTSON R M, WESTWOOD J T, WALKER V K. Cold hardening and transcriptional change in Drosophila melanogaster. Insect Molecular Biology, 2005, 14(6): 607-613.

[26] HUANG L H, KANG L. Cloning and interspecific altered expression of heat shock protein genes in two leafminer species in response to thermal stress. Insect Molecular Biology, 2007, 16(4): 491-500.

[27] YOSHIMI T, MINOWA K, KAROUNA-RENIER N K, WATANABE C, SUGAYA Y, MIURA T. Activation of a stress- induced gene by insecticides in the midge, Chironomus yoshimatsui. Journal of Biochemical and Molecular Toxicology, 2002, 16(1): 10-17.

[28] LIN Q S, JIN F L, HU Z D, CHEN H Y, YIN F, LI Z Y, DONG X L, ZHANG D Y, REN S X, FENG X. Transcriptome analysis of chlorantraniliprole resistance development in the diamondback moth Plutella xylostella. PLoS ONE, 2013, 8(8): e72314.

[29] WAN P J, GUO W Y, YANG Y, LU F G, LU W P, LI G Q. RNAi suppression of the ryanodine receptor gene results in decreased susceptibility to chlorantraniliprole in Colorado potato beetle Leptinotarsa decemlineata. Journal of Insect Physiology, 2014, 63(1): 48-55.

[30] YANG Y, WAN P J, HU X X, LI G Q. RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furcifera. Pesticide Biochemistry and Physiology, 2014, 108: 58-65.