表皮蛋白基因参与锈赤扁谷盗磷化氢抗性形成

doi:10.3864/j.issn.0578-1752.2023.09.007

摘要/Abstract

摘要：

【目的】作为昆虫表皮重要的结构物质，表皮蛋白（cuticle protein，CP）在昆虫对农药表皮穿透抗性形成过程中承担重要作用。锈赤扁谷盗（Cryptolestes ferrugineus）磷化氢抗性问题日益突出，论文旨在揭示表皮蛋白基因在该害虫磷化氢抗性形成过程中的作用。【方法】基于联合国粮农组织（FAO）推荐的生物测定方法解析5个地理种群（张家港、湘阴、淮安、怀化和太仓种群)锈赤扁谷盗磷化氢敏感性差异。首先通过锈赤扁谷盗转录组数据鉴定获得4个表皮蛋白基因，进而构建系统发育树，并对相应氨基酸序列进行分析。利用基因定量技术（RT-qPCR）解析上述4个锈赤扁谷盗表皮蛋白基因时空（不同发育阶段和成虫不同组织）和不同磷化氢抗性水平下的表达模式，以及表皮蛋白基因对磷化氢胁迫响应的表达模式。使用RNAi（RNA interference）技术对特定的表皮蛋白基因（CfRR2-1）进行沉默，并研究CfRR2-1被有效沉默后锈赤扁谷盗磷化氢敏感性变化情况。【结果】磷化氢敏感性测定结果显示，不同地理种群锈赤扁谷盗磷化氢敏感性水平差异显著，药剂抗性倍数（RR）范围为7.2—1 906.8。系统发育分析显示锈赤扁谷盗4个表皮蛋白均隶属于CPR家族RR2亚家族，且它们氨基酸序列都拥有RR2型几丁质结合域，将其分别命名为CfRR2-1、CfRR2-2、CfRR2-3和CfRR2-4。基因表达模式分析显示4个表皮蛋白基因均在锈赤扁谷盗蛹期特异性高表达，且在成虫外周组织中表达水平较高；此外，表皮蛋白基因均在磷化氢极高抗种群中（太仓种群，RR=1 906.8）显著高表达，且锈赤扁谷盗经磷化氢熏蒸胁迫后，表皮蛋白基因可被显著诱导表达。最后，选择CfRR2-1进行功能验证，注射dsRNA干扰极高抗种群（TC）CfRR2-1的表达后，导致锈赤扁谷盗对磷化氢抗性水平显著降低。【结论】表皮蛋白基因过表达参与锈赤扁谷盗磷化氢抗性形成。

关键词: 锈赤扁谷盗, 磷化氢抗性, 表皮蛋白, RNA干扰

Abstract:

【Objective】As an important structural component of insect cuticle, the cuticle protein (CP) plays an important role in the formation of cuticle penetration resistance to pesticides. The phosphine resistance of Cryptolestes ferrugineus is increasingly prominent, and the current study was conducted to reveal the roles of CP genes in the formation of phosphine resistance in C. ferrugineus.【Method】According to the phosphine bioassay method that recommended by the Food and Agriculture Organization of the United Nations (FAO), the difference in phosphine sensitivity from five geographical populations (Zhangjiagang, Xiangyin, Huaian, Huaihua and Taicang populations) of C. ferrugineus was analyzed. The four CP genes were identified from the previous transcriptome data of C. ferrugineus, and then the phylogenetic tree of CPs was constructed and the corresponding amino acid sequence of C. ferrugineus CPs was further analyzed. Afterwards, the RT-qPCR was used to analyze the spatio-temporal (different developmental stages and different tissues of adults) expression patterns of four CP genes, and their expression levels under different phosphine resistance levels, as well as the expression patterns of four CP genes in response to phosphine stress were explored. Subsequently, a specific CP gene (CfRR2-1) was selected to be knocked down by using RNAi (RNA interference) technology, and the change of phosphine sensitivity of C. ferrugineus was determined.【Result】The results of phosphine sensitivity bioassay analysis showed that there were significant differences in phosphine resistance levels of different geographical populations, and the range of insecticide resistance ratio (RR) was 7.2-1 906.8. The further sequence analysis suggested that the four CPs all contained chitin binding domain, which belonged to the RR2 subfamily of CPR family, and they were named as CfRR2-1, CfRR2-2, CfRR2-3 and CfRR2-4, respectively. The gene expression patterns demonstrated that four CP genes were specifically highly expressed in the pupal stage of C. ferrugineus, and the high expression levels of four CP genes were detected in the peripheral tissues of C. ferrugineus as well. Besides, the CP genes were highly expressed in the phosphine resistant population (Taicang population, RR=1 906.8), and their expression levels could be significantly induced by phosphine in C. ferrugineus. Lastly, a CP gene CfRR2-1 was selected for the further functional study. After the gene expression level of CfRR2-1 was significantly knocked down in phosphine resistance (TC) population of C. ferrugineus via the injection of dsRNA, the sensitivity of C. ferrugineus to phosphine was significantly increased.【Conclusion】The over-expression of CP gene is involved in the formation of phosphine resistance.

Key words: Cryptolestes ferrugineus, phosphine resistance, cuticle protein, RNA interference (RNAi)

陈二虎, 沈丹蓉, 杜文蔚, 孟宏杰, 唐培安. 表皮蛋白基因参与锈赤扁谷盗磷化氢抗性形成[J]. 中国农业科学, 2023, 56(9): 1696-1707.

CHEH ErHu, SHEN DanRong, DU WenWei, MENG HongJie, TANG PeiAn. Cuticle Protein Genes are Involved in Phosphine Resistance of Cryptolestes ferrugineus[J]. Scientia Agricultura Sinica, 2023, 56(9): 1696-1707.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.09.007

https://www.chinaagrisci.com/CN/Y2023/V56/I9/1696

图/表 8

表1

表2

图1

图2

图3

图4

图5

图6

参考文献 45

[1]	PHILLIPS T W, THRONE J E. Biorational approaches to managing stored-product insects. Annual Review of Entomology, 2010, 55: 375-397. doi: 10.1146/annurev.ento.54.110807.090451 pmid: 19737083
[2]	LOSEY S M, DAGLISH G J, PHILLIPS T W. Orientation of rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), to semiochemicals in field and laboratory experiments. Journal of Stored Products Research, 2019, 84: 101513. doi: 10.1016/j.jspr.2019.101513
[3]	AULICKY R, STEJSKAL V, FRYDOVA B. Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic. Journal of Stored Products Research, 2019, 81: 107-113. doi: 10.1016/j.jspr.2019.02.003
[4]	NAYAK M K, HOLLOWAY J C, EMERY R N, PAVIC H, BARTLET J, COLLINS P J. Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): Its characterisation, a rapid assay for diagnosis and its distribution in Australia. Pest Management Science, 2013, 69(1): 48-53. doi: 10.1002/ps.2013.69.issue-1
[5]	AGRAFIOTI P, ATHANASSIOU C G, NAYAK M K. Detection of phosphine resistance in major stored-product insects in Greece and evaluation of a field resistance test kit. Journal of Stored Products Research, 2019, 82: 40-47. doi: 10.1016/j.jspr.2019.02.004
[6]	NAYAK M K, DAGLISH G J, PHILLIPS T W, EBERT P R. Resistance to the fumigant phosphine and its management in insect pests of stored products: A global perspective. Annual Review of Entomology, 2020, 65: 333-350. doi: 10.1146/annurev-ento-011019-025047 pmid: 31610132
[7]	ALZAHRANI S, EBERT P R. Oxygen and arsenite synergize phosphine toxicity by distinct mechanisms. Toxicological Sciences, 2019, 167(2): 419-425. doi: 10.1093/toxsci/kfy248 pmid: 30304530
[8]	ZURYN S, KUANG J, EBERT P. Mitochondrial modulation of phosphine toxicity and resistance in Caenorhabditis elegans. Toxicological Sciences, 2008, 102(1): 179-186. doi: 10.1093/toxsci/kfm278
[9]	PIMENTEL M A G, FARONI L R D, TÓTOLA M R, GUEDES R N C. Phosphine resistance, respiration rate and fitness consequences in stored-product insects. Pest Management Science, 2007, 63(9): 876-881. pmid: 17597470
[10]	OPIT G P, PHILLIPS T W, AIKINS M J, HASAN M M. Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. Journal of Economic Entomology, 2012, 105(4): 1107-1114. doi: 10.1603/EC12064
[11]	SCHLIPALIUS D I, VALMAS N, TUCK A G, JAGADEESAN R, MA L, KAUR R, GOLDINGER A, ANDERSON C, KUANG J, ZURYN S, et al. A core metabolic enzyme mediates resistance to phosphine gas. Science, 2012, 338(6108): 807-810. doi: 10.1126/science.1224951 pmid: 23139334
[12]	SCHLIPALIUS D I, TUCK A G, PAVIC H, DAGLISH G, NAYAK M K, EBERT P R. A high-throughput system used to determine frequency and distribution of phosphine resistance across large geographical regions. Pest Management Science, 2019, 75(4): 1091-1098. doi: 10.1002/ps.5221 pmid: 30255667
[13]	HUANG Y, LI F F, LIU M W, WANG Y Z, SHEN F, TANG P A. Susceptibility of Tribolium castaneum to phosphine in China and functions of cytochrome P450s in phosphine resistance. Journal of Pest Science, 2019, 92: 1239-1248. doi: 10.1007/s10340-019-01088-7
[14]	YANG J O, PARK J S, LEE H S, KWON M, KIM G H, KIM J. Identification of a phosphine resistance mechanism in Rhyzopertha dominica based on transcriptome analysis. Journal of Asia-Pacific Entomology, 2018, 21(4): 1450-1456. doi: 10.1016/j.aspen.2018.11.012
[15]	GIROTTI J R, MIJAILOVSKY S J, PATRICIA JUÁREZ M. Epicuticular hydrocarbons of the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). Physiological Entomology, 2012, 37(3): 266-277. doi: 10.1111/pen.2012.37.issue-3
[16]	MOUSSIAN B. Recent advances in understanding mechanisms of insect cuticle differentiation. Insect Biochemistry and Molecular Biology, 2010, 40(5): 363-375. doi: 10.1016/j.ibmb.2010.03.003 pmid: 20347980
[17]	BALABANIDOU V, GRIGORAKI L, VONTAS J. Insect cuticle: A critical determinant of insecticide resistance. Current Opinion in Insect Science, 2018, 27: 68-74. doi: S2214-5745(17)30119-0 pmid: 30025637
[18]	CHEN N, PEI X J, LI S, FAN Y L, LIU T X. Involvement of integument-rich CYP4G19 in hydrocarbon biosynthesis and cuticular penetration resistance in Blattella germanica (L.). Pest Management Science, 2020, 76(1): 215-226. doi: 10.1002/ps.v76.1
[19]	DANG K, DOGGETT S L, SINGHAM G V, LEE C Y. Insecticide resistance and resistance mechanisms in bed bugs, Cimex spp. (Hemiptera: Cimicidae). Parasites & Vectors, 2017, 10(1): 318.
[20]	YANG C H, YANG P C, ZHANG S F, SHI Z Y, KANG L, ZHANG A B. Identification, expression pattern, and feature analysis of cuticular protein genes in the pine moth Dendrolimus punctatus (Lepidoptera: Lasiocampidae). Insect Biochemistry and Molecular Biology, 2017, 83: 94-106. doi: 10.1016/j.ibmb.2017.03.003
[21]	刘晓健, 刘卫敏, 赵小明, 张建珍, 马恩波. 昆虫表皮发育研究进展及展望. 应用昆虫学报, 2019, 56(4): 625-638.
	LIU X J, LIU W M, ZHAO X M, ZHANG J Z, MA E B. Progress in the study of insect cuticle development and prospects for future research. Chinese Journal of Applied Entomology, 2019, 56(4): 625-638. (in Chinese)
[22]	QIAO L, XIONG G, WANG R X, HE S Z, CHEN J, TONG X L, HU H, LI C L, GAI T T, XIN Y Q, LIU X F, CHEN B, XIANG Z H, LU C, DAI F Y. Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori. Genetics, 2014, 196(4): 1103-1115. doi: 10.1534/genetics.113.158766 pmid: 24514903
[23]	ASANO T, TAOKA M, SHINKAWA T, YAMAUCHI Y, ISOBE T, SATO D. Identification of a cuticle protein with unique repeated motifs in the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology, 2013, 43(4): 344-351. doi: 10.1016/j.ibmb.2013.01.001 pmid: 23376333
[24]	陈二虎, 孟宏杰, 陈艳, 唐培安. 表皮蛋白基因TcCP14.6和TcLCPA3A参与介导赤拟谷盗对磷化氢的抗性形成. 中国农业科学, 2022, 55(11): 2150-2160. doi: 10.3864/j.issn.0578-1752.2022.11. 006. doi: 10.3864/j.issn.0578-1752.2022.11.006
	CHEN E H, MENG H J, CHEN Y, TANG P A. Cuticle protein genes TcCP14.6 and TcLCPA3A are involved in phosphine resistance of Tribolium castaneum. Scientia Agricultura Sinica, 2022, 55(11): 2150-2160. doi: 10.3864/j.issn.0578-1752.2022.11.006. (in Chinese) doi: 10.3864/j.issn.0578-1752.2022.11.006
[25]	TAMURA K, STECHER G, PETERSON D, FILIPSKI A, KUMAR S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 2013, 30(12): 2725-2729. doi: 10.1093/molbev/mst197 pmid: 24132122
[26]	TANG P A, DUAN J Y, WU H J, JU X R, YUAN M L. Reference gene selection to determine differences in mitochondrial gene expressions in phosphine-susceptible and phosphine-resistant strains of Cryptolestes ferrugineus, using qRT-PCR. Scientific Reports, 2017, 7: 7047. doi: 10.1038/s41598-017-07430-2
[27]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔ^C^Tmethod. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262
[28]	KAUR R, SUBBARAYALU M, JAGADEESAN R, DAGLISH G J, NAYAK M K, NAIK H R, RAMASAMY S, SUBRAMANIAN C, EBERT P R, SCHLIPALIUS D I. Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes. Heredity, 2015, 115(3): 188-194. doi: 10.1038/hdy.2015.24 pmid: 25853517
[29]	KONEMANN C E, HUBHACHEN Z, OPIT G P, GAUTAM S, BAJRACHARYA N S. Phosphine resistance in Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) collected from grain storage facilities in Oklahoma, USA. Journal of Economic Entomology, 2017, 110(3): 1377-1383.
[30]	PRICE N R. Active exclusion of phosphine as a mechanism of resistance in Rhyzopertha dominica (F.)(Coleoptera: Bostrychidae). Journal of Stored Products Research, 1984, 20(3): 163-168. doi: 10.1016/0022-474X(84)90025-0
[31]	OPPERT B, GUEDES R N C, AIKINS M J, PERKIN L, CHEN Z, PHILLIPS T W, ZHU K Y, OPIT G P, HOON K, SUN Y, et al. Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum. BMC Genomics, 2015, 16: 968. doi: 10.1186/s12864-015-2121-0
[32]	TANG L, LIANG J, ZHAN Z, XIANG Z, HE N. Identification of the chitin-binding proteins from the larval proteins of silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology, 2010, 40(3): 228-234. doi: 10.1016/j.ibmb.2010.01.010 pmid: 20149871
[33]	叶长青, 包涵, 刘田, 杨青. 双叉犀金龟表皮蛋白TdCPR12611与TdCPR7854的表达纯化及特性分析. 昆虫学报, 2021, 64(1): 19-29.
	YE C Q, BAO H, LIU T, YANG Q. Expression, purification and characterization of the cuticular proteins TdCPR12611 and TdCPR7854 from Trypoxylus dichotomus (Coleoptera: Scarabaeidae). Acta Entomologica Sinica, 2021, 64(1): 19-29. (in Chinese)
[34]	VANNINI L, WILLIS J H. Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae. Arthropod Structure and Development, 2017, 46(1): 13-29. doi: 10.1016/j.asd.2016.10.002
[35]	SHAHIN R, IWANAGA M, KAWASAKI H. Cuticular protein and transcription factor genes expressed during prepupal-pupal transition and by ecdysone pulse treatment in wing discs of Bombyx mori. Insect Molecular Biology, 2016, 25(2): 138-152. doi: 10.1111/imb.2016.25.issue-2
[36]	丛林, 刘浩强, 李鸿筠, 巴音克西克, 冉春. 褐色橘蚜RR-2型表皮蛋白基因鉴定及功能分析. 植物保护学报, 2020, 47(5): 1078-1087.
	CONG L, LIU H Q, LI H J, BAYINKEXIKE, RAN C. Identification and function analysis of RR-2 CPR genes in brown citrus aphid Toxoptera citricida (Kirkaldy). Journal of Plant Protection, 2020, 47(5): 1078-1087. (in Chinese)
[37]	LIANG J, WANG T, XIANG Z, HE N. Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli. Insect Biochemistry and Molecular Biology, 2015, 58: 76-88. doi: 10.1016/j.ibmb.2014.11.004
[38]	SILVA A X, BACIGALUPE L D, LUNA-RUDLOFF M, FIGUEROA C C. Insecticide resistance mechanisms in the green peach aphid Myzus persicae (Hemiptera: Aphididae) II: Costs and benefits. PLoS ONE, 2012, 7(6): e36810. doi: 10.1371/journal.pone.0036810
[39]	ZHOU D, DUAN B, SUN Y, MA L, ZHU C, SHEN B. Preliminary characterization of putative structural cuticular proteins in the malaria vector Anopheles sinensis. Pest Management Science, 2017, 73(12): 2519-2528. doi: 10.1002/ps.2017.73.issue-12
[40]	SUN X, GUO J, YE W, GUO Q, HUANG Y, MA L, ZHOU D, SHEN B, SUN Y, ZHU C. Cuticle genes CpCPR63 and CpCPR47 may confer resistance to deltamethrin in Culex pipiens pallens. Parasitology Research, 2017, 116(8): 2175-2179. doi: 10.1007/s00436-017-5521-z
[41]	HUANG Y, GUO Q, SUN X H, ZHANG C, XU N, XU Y, ZHOU D, SUN Y, MA L, ZHU C L, SHEN B. Culex pipiens pallens cuticular protein CPLCG5 participates in pyrethroid resistance by forming a rigid matrix. Parasites & Vectors, 2018, 11: 6.
[42]	YAHOUÉDO G A, CHANDRE F, ROSSIGNOL M, GINIBRE C, BALABANIDOU V, MENDEZ N G A, PIGEON O, VONTAS J, CORNELIE S. Contributions of cuticle permeability and enzyme detoxification to pyrethroid resistance in the major malaria vector Anopheles gambiae. Scientific Reports, 2017, 7: 11091. doi: 10.1038/s41598-017-11357-z
[43]	KOGANEMARU R, MILLER D M, ADELMAN Z N. Robust cuticular penetration resistance in the common bed bug (Cimex lectularius L.) correlates with increased steady-state transcript levels of CPR-type cuticle protein genes. Pesticide Biochemistry and Physiology, 2013, 106(3): 190-197. doi: 10.1016/j.pestbp.2013.01.001
[44]	张万娜, 刘香亚, 赖乾, 肖海军. 棉铃虫表皮蛋白基因CP22和CP14的表达特征及其对甲氧虫酰肼的响应. 植物保护学报, 2021, 48(5): 1043-1053.
	ZHANG W N, LIU X Y, LAI Q, XIAO H J. Expression analysis of cuticular protein genes CP22 and CP14 in cotton bollworm Helicoverpa armigera and their response to the sublethal dose of methoxyfenozide. Journal of Plant Protection, 2021, 48(5): 1043-1053. (in Chinese)
[45]	CHEN E H, HOU Q L, DOU W, WEI D D, YUE Y, YANG R L, YANG P J, YU S F, DE SCHUTTER K, SMAGGHE G, WANG J J. Genome-wide annotation of cuticular proteins in the oriental fruit fly (Bactrocera dorsalis), changes during pupariation and expression analysis of CPAP3 protein genes in response to environmental stresses. Insect Biochemistry Molecular Biology, 2018, 97: 53-70. doi: 10.1016/j.ibmb.2018.04.009

引物类型Primer type	引物名称Primer name	引物序列Primer sequence (5′to 3′)
qPCR引物 Primers for qPCR	CfRR2-1-F	CGGACACACTGGAGACAAGA
	CfRR2-1-R	TCCATGAGTGGCTACGACAG
	CfRR2-2-F	CACCGTCAAAGGCCAATACT
	CfRR2-2-R	ATGACCAAGACCACCGAGTC
	CfRR2-3-F	TACCATACCCCAGCCCATTA
	CfRR2-3-R	TACAGCATGTCCCACACGTT
	CfRR2-4-F	TGCAGGCATTCTTGGATATG
	CfRR2-4-R	ACCATCAGGTTCAGCTACGG
	CfRPS13-F	ATCCGTAAGCATTTGGAACG
	CfRPS13-R	AGCCACTAAGGCTGAAGCTG
	CfEFLα-F	CCAGGCATGGTAGTGACCTT
	CfEFLα-R	TTGGAGGGTTGTTTTTGGAG
dsRNA引物 Primers for dsRNA	dsCfRR2-1-F	ggatcctaatacgactcactataggGTGATTGGAGGCGGAATTGG
	dsCfRR2-1-R	ggatcctaatacgactcactataggTCCACCTCCCAATCCAAGTC
	dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
	dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA

种群 Population	采集地 Collection site	抗性倍数 Resistance ratio
ZJG	张家港Zhangjiagang	7.2
XY	湘阴 Xiangyin	29.6
HA	淮安Huaian	325.4
HH	怀化Huaihua	362.7
TC	太仓Taicang	1906.8