二化螟实时荧光定量PCR内参基因筛选和表达稳定性评价
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摘要
【目的】筛选特定试验条件下二化螟(Chilosuppressalis)稳定表达的内参基因,为二化螟基因表达研究奠定基础。【方法】根据二化螟转录组测序结果挑选出11个候选基因,利用实时荧光定量PCR测定其在二化螟不同发育历期、不同组织、温度处理、杀虫剂处理、取食不同饲料、取食不同水稻、dsRNA处理和混合样品的表达量,利用RefFinder、BestKeeper、Ge Norm和NormFinder等软件和ΔC_t值对11个候选基因的稳定性进行评估。【结果】5种分析方法表明,在二化螟不同发育历期稳定性较高的内参基因是AK、RPL10和EF1,不同组织中稳定性较高的内参基因是EF1、TUB和ACTB,不同温度下较稳定的内参基因为TUB、RPL10和EF1,杀虫剂处理样品中较稳定的是TF4和ACTA,取食不同饲料较稳定的是TUB、TF4、EF1和RPL10,取食不同品种水稻较稳定的是TUB和EF1,dsRNA处理样品中较稳定的是TUB、AK、ACTB和EF1,混合样品中较稳定的是EF1、TUB和ACTB。【结论】为不同试验条件下选择合适的内参基因提供了参考,也有利于在二化螟基因表达研究中获得更加可靠准确的数据。
【Objective】We aim to screen reference genes stably expressed in striped stem borers(SSB), Chilo suppressalis, under certain conditions, to lay a foundation for the study on the gene expression in SSB. 【Method】According to the results of SSB transcriptome data, 11 candidate genes from SSB were selected and their expression stability was detected under different treatment conditions(including development duration, tissues, feeding on different diets and rice varieties, dsRNA and mixed samples under temperature stress, insecticide treatment), by using real-time fluorescence quantitative PCR. The stability of the 11 candidate genes was evaluated by RefFinder, BestKeeper, GeNorm, NormFinder and ΔC_(t~.) 【Result】AK, RPL10 and EF1 were highly stable in different development durations; EF1, TUB and ACTB were highly stable in different tissues. TUB, RPL10 and EF1 were relatively stable under temperature stress. TF4 and ACTA were stable in insecticide treated samples. TUB, TF4, EF1 and RPL10 were stable in samples fed on different diets. TUB and EF1 were stable reference genes in samples fed on different rice varieties. TUB, AK, ACTB and EF1 were stable in the sample treated with dsRNA. EF1, TUB and ACTB were stable in mixed samples. 【Conclusion】 It provide a reference for selection of suitable reference gene under different test conditions, and also provide more reliable and accurate data for research in gene expression in rice SSB.
【Objective】We aim to screen reference genes stably expressed in striped stem borers(SSB), Chilo suppressalis, under certain conditions, to lay a foundation for the study on the gene expression in SSB. 【Method】According to the results of SSB transcriptome data, 11 candidate genes from SSB were selected and their expression stability was detected under different treatment conditions(including development duration, tissues, feeding on different diets and rice varieties, dsRNA and mixed samples under temperature stress, insecticide treatment), by using real-time fluorescence quantitative PCR. The stability of the 11 candidate genes was evaluated by RefFinder, BestKeeper, GeNorm, NormFinder and ΔC_(t~.) 【Result】AK, RPL10 and EF1 were highly stable in different development durations; EF1, TUB and ACTB were highly stable in different tissues. TUB, RPL10 and EF1 were relatively stable under temperature stress. TF4 and ACTA were stable in insecticide treated samples. TUB, TF4, EF1 and RPL10 were stable in samples fed on different diets. TUB and EF1 were stable reference genes in samples fed on different rice varieties. TUB, AK, ACTB and EF1 were stable in the sample treated with dsRNA. EF1, TUB and ACTB were stable in mixed samples. 【Conclusion】 It provide a reference for selection of suitable reference gene under different test conditions, and also provide more reliable and accurate data for research in gene expression in rice SSB.
引文
[1]Bustin S A.Absolute quantification of m RNA using real-time reverse transcription polymerase chain reaction assays.J Mol Endocrinol,2000,25:169-193.
[2]Thellin O,Zorzi W,Lakaye B,De Borman B,Coumans B,Hennen G,Grisar T,Igout A,Heinen E.Housekeeping genes as internal standards:Use and limits.J Biotechnol,1999,75:291-295.
[3]de Jonge H J,Fehrmann R S,de Bont E S,Hofstra R M,Gerbens F,Kamps W A,de Vries E G,van der Zee A G,te Meerman G J,ter Elst A.Evidence based selection of housekeeping genes.PLoS One,2007,2:e898.
[4]Gutierrez L,Mauriat M,Pelloux J,Bellini C,Van Wuytswinkel O.Towards a systematic validation of references in real-time RT-PCR.Plant Cell,2008,20:1734-1735.
[5]Huggett J,Dheda K,Bustin S,Zumla A.Real-time RT-PCR normalisation;strategies and considerations.Genes Immun,2005,6:279-284.
[6]Vandesompele J,De Preter K,Pattyn F,Poppe B,Van Roy N,De Paepe A,Speleman F.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,2002,3:Research0034.
[7]符伟,谢文,张卓,吴青君,王少丽,张友军.Bt毒素诱导下小菜蛾实时定量PCR内参基因的筛选.昆虫学报,2012,55(12):1406-1412.Fu W,Xie W,Zhang Z,Wu Q J,Wang S L,Zhang Y J.Selection of valid reference genes for gene expression studies by quantitative real-time PCR in Plutella xylostella(Lepidoptera:Plutellidae)after exposure to Bt toxin.Acta Entomol Sin,2012,55(12):1406-1412.(in Chinese with English abstract)
[8]Lu Y H,Yuan M,Gao X W,Kang T H,Zhan S,Wan H,Li J H.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLoS One,2013,8:e68059.
[9]Chandra GS,Asokan R,Manamohan M,Kumar N K,Sita T.Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm,Helicoverna armigera.Mol Biol,2014,48:927-938.
[10]Shakeel M,Zhu X,Kang T,Wan H,Li J.Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm,Helicoverpa armigera(Lepidoptera:Noctuidae).J Asia-Pac Entomol,2015,18:123-130.
[11]Zhang S D,An S H,Li Z,Wu F M,Yang Q P,Liu Y C,Cao J J,Zhang H J,Zhang Q W,Liu X X.Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae).Gene,2015,555:393-402.
[12]Zhu X,Yuan M,Shakeel M,Zhang Y J,Wang S L,Wang X,Zhan S,Kang T H,Li J H.Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua(Hubner)(Lepidoptera:Noctuidae).PLoS One,2014,9:e84730.
[13]Arun A,Baumle V,Amelot G,Nieberding C M.Selection and validation of reference genes for qRT-PCRexpression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.PLoS One,2015,10:e0120401.
[14]Lu Y H,Zheng X S,Liang Q,Xu H X,Yang Y J,Tian JC,He X C,Lu Z X.Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens(Lepidoptera:Noctuidae).J Asia-Pac Entomol,2015,18:669-675.
[15]Sun M,Lu M X,Tang X T,Du Y Z.Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens(Lepidoptera:Noctuidae).PLoS One,2015,10:e0115979.
[16]Pan H P,Yang X W,Bidne K,Hellmich R L,Siegfried BD,Zhou X G.Selection of reference genes for RT-qPCRanalysis in the monarch butterfly,Danaus plexippus(L.),a migrating bio-Indicator.PLoS One,2015,10:e0129482.
[17]Liu G Q,Qiu X H,Cao L,Zhang Y,Zhan Z B,Han R C.Evaluation of reference genes for reverse transcription quantitative PCR studies of physiological responses in the ghost moth,Thitarodes armoricanus(Lepidoptera,Hepialidae).PLoS One,2016,11:e0159060.
[18]Zhang L,Zhang Q L,Wang X T,Yang X Z,Li X P,Yuan M L.Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars(Lepidoptera:Erebidae:Gynaephora)along an altitude gradient.Ecol Evol,2017,7:9054-9065.
[19]杨苓,胡晓静,徐志峰,何林,肖伟.桃蛀螟实时荧光定量PCR内参基因的筛选.昆虫学报,2017,60(11):1266-1277.Yang L,Hu X J,Xu Z F,He L,Xiao W.Screening of reference genes for qRT-PCR in Conogethes punctiferails(Lepidoptera:Crambidae).Acta Entomol Sin,2017,60(11):1266-1277.(in Chinese with English abstract)
[20]Teng X L,Zhang Z,He G L,Yang L W,Li F.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.J Insect Sci,2011,12:60.
[21]Xu J,Lu M X,Cui Y D,Du Y Z.Selection and evaluation of reference genes for expression analysis using qRT-PCR in Chilo suppressalis(Lepidoptera:Pyralidae).J Econ Entomol,2017,110:683-691.
[22]胡阳,郑永利,曹国连,傅强.利用半人工饲料大规模简便化饲养二化螟中国水稻科学,2013,27(5):535-538.Hu Y,Zheng Y L,Cao G L,Fu Q.A technique for rearing Chilo suppressalis in the large scale with an oligidic diet in laboratory.Chin J Rice Sci,2013,27(5):535-538.(in Chinese with English abstract)
[23]吴敏,张真真,高聪芬.水稻二化螟抗药性监测方法.应用昆虫学报,2013,50(2):548-552.Wu M,Zhang Z Z,Gao C F.Methods of insecticides resistance monitoring of the striped stem borer,Chilo suppressalis.Chin J Appl Entomol,2013,50(2):548-552.(in Chinese with English abstract)
[24]Hui X M,Yang W,He G L,Yang Q P,Han Z,Li F.RNAinterference of ace1 and ace2 in Chilo suppressalis reveals their different contributions to motor ability and larval growth.Insect Mol Biol,2011,4:507-518.
[25]Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity.BestKeeper:Excel-based tool using pair-wise correlations.Biotechnol Lett,2004,26:509-515.
[26]Andersen C L,Jensen J L,Orntoft T F.Normalization of real-time quantitative reverse transcription-PCR data:Amodel-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Res,2004,64:5245-5250.
[27]Schmittgen T D,Livak K J.Analyzing real-time PCRdata by the comparative CT method.Nat Protoc,2008,3:1101-1108.
[28]Radonic A,Thulke S,Mackay I M,Landt O,Siegert W,Nitsche A.Guideline to reference gene selection for quantitative real-time PCR.Biochem Biophys Res Commun,2004,313:856-862.
[29]Huis R,Hawkins S,Neutelings G.Selection of reference genes for quantitative gene expression normalization in flax(Linum usitatissimum L.).BMC Plant Biol,2010,10:71.
[30]Kozera B,Rapacz M.Reference genes in real-time PCR.J Appl Genet,2013,54:391-406.
[31]冯波,郭前爽,毛必鹏,杜永均.松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选.昆虫学报,2016,59(4):427-437.Feng B,Guo Q,Mao B P,Du Y J.Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus(Coleoptera:Cerambycidae)by RT-PCR.Acta Entomol Sin,2016,59(4):427-437.(in Chinese with English abstract)
[32]Nielsen M G,Gadagkar S R,Gutzwiller L.Tubulin evolution in insects:Gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family.BMC Evol Biol,2010,10:113.
[33]Yuan M,Lu Y H,Zhu X,Wan H,Shakeel M,Zhan S,Jin B R,Li J H.Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)using reverse-transcription quantitative PCR.PLoS One,2014,9:e86503.
[34]An X K,Hou M L,Liu Y D.Reference gene selection and evaluation for gene expression studies using qRT-PCR in the white-backed planthopper,Sogatella furcifera(Hemiptera:Delphacidae).J Econ Entomol,2016,109:879-886.
[35]Dai T M,Lu Z C,Liu W X,Wan F H.Selection and validation of reference genes for qRT-PCR analysis during biological invasions:The thermal adaptability of Bemisia tabaci MED.PLoS One,2017,12:e0173821.
[36]Ponton F,Chapuis M P,Pernice M,Sword GA,Simpson S J.Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster.J Insect Physiol,2011,57:840-850.
[37]Yang Q P,Li Z,Cao J J,Zhang S D,Zhang H J,Wu X Y,Zhang Q W,Liu X X.Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria(Orthoptera:Acrididae).PLoS One,2014,9:e98164.
[38]Zheng Y T,Li H B,Lu M X,Du Y Z.Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis(Thysanoptera:Thripidae).PLoS One,2014,9:e111369.
[39]Pan H P,Yang X W,Siegfried B D,Zhou X G.Acomprehensive selection of reference genes for RT-qPCRanalysis in a predatory lady beetle,Hippodamia convergens(Coleoptera:Coccinellidae).PLoS One,2015,10:e0125868.
[40]Qiu L,Wang P,Wu T,Li B,Wang X,Lei C,Lin Y,Zhao J,Ma W.Downregulation of Chilo suppressalis alkaline phosphatase genes associated with resistance to three transgenic Bacillus thuringiensis rice lines.Insect Mol Biol,2018,27:83-89.
[41]Xu G,Gu G X,Teng Z W,Wu S F,Huang J,Song Q S,Ye G Y,Fang Q.Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.Sci Rep,2016,6:28976.
[42]Xu G,Wu S F,Teng Z W,Yao H W,Fang Q,Huang J,Ye G Y.Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer,Chilo suppressalis(Lepidoptera:Crambidae).Insect Sci,2017,24:371-384.
[43]Xu G,Wu S F,Wu Y S,Gu G X,Fang Q,Ye G Y.De novo assembly and characterization of central nervous system transcriptome reveals neurotransmitter signaling systems in the rice striped stem borer,Chilo suppressalis.BMC Genomics,2015,16:525.
[2]Thellin O,Zorzi W,Lakaye B,De Borman B,Coumans B,Hennen G,Grisar T,Igout A,Heinen E.Housekeeping genes as internal standards:Use and limits.J Biotechnol,1999,75:291-295.
[3]de Jonge H J,Fehrmann R S,de Bont E S,Hofstra R M,Gerbens F,Kamps W A,de Vries E G,van der Zee A G,te Meerman G J,ter Elst A.Evidence based selection of housekeeping genes.PLoS One,2007,2:e898.
[4]Gutierrez L,Mauriat M,Pelloux J,Bellini C,Van Wuytswinkel O.Towards a systematic validation of references in real-time RT-PCR.Plant Cell,2008,20:1734-1735.
[5]Huggett J,Dheda K,Bustin S,Zumla A.Real-time RT-PCR normalisation;strategies and considerations.Genes Immun,2005,6:279-284.
[6]Vandesompele J,De Preter K,Pattyn F,Poppe B,Van Roy N,De Paepe A,Speleman F.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,2002,3:Research0034.
[7]符伟,谢文,张卓,吴青君,王少丽,张友军.Bt毒素诱导下小菜蛾实时定量PCR内参基因的筛选.昆虫学报,2012,55(12):1406-1412.Fu W,Xie W,Zhang Z,Wu Q J,Wang S L,Zhang Y J.Selection of valid reference genes for gene expression studies by quantitative real-time PCR in Plutella xylostella(Lepidoptera:Plutellidae)after exposure to Bt toxin.Acta Entomol Sin,2012,55(12):1406-1412.(in Chinese with English abstract)
[8]Lu Y H,Yuan M,Gao X W,Kang T H,Zhan S,Wan H,Li J H.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLoS One,2013,8:e68059.
[9]Chandra GS,Asokan R,Manamohan M,Kumar N K,Sita T.Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm,Helicoverna armigera.Mol Biol,2014,48:927-938.
[10]Shakeel M,Zhu X,Kang T,Wan H,Li J.Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm,Helicoverpa armigera(Lepidoptera:Noctuidae).J Asia-Pac Entomol,2015,18:123-130.
[11]Zhang S D,An S H,Li Z,Wu F M,Yang Q P,Liu Y C,Cao J J,Zhang H J,Zhang Q W,Liu X X.Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae).Gene,2015,555:393-402.
[12]Zhu X,Yuan M,Shakeel M,Zhang Y J,Wang S L,Wang X,Zhan S,Kang T H,Li J H.Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua(Hubner)(Lepidoptera:Noctuidae).PLoS One,2014,9:e84730.
[13]Arun A,Baumle V,Amelot G,Nieberding C M.Selection and validation of reference genes for qRT-PCRexpression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.PLoS One,2015,10:e0120401.
[14]Lu Y H,Zheng X S,Liang Q,Xu H X,Yang Y J,Tian JC,He X C,Lu Z X.Evaluation and validation of reference genes for SYBR Green qRT-PCR normalization in Sesamia inferens(Lepidoptera:Noctuidae).J Asia-Pac Entomol,2015,18:669-675.
[15]Sun M,Lu M X,Tang X T,Du Y Z.Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens(Lepidoptera:Noctuidae).PLoS One,2015,10:e0115979.
[16]Pan H P,Yang X W,Bidne K,Hellmich R L,Siegfried BD,Zhou X G.Selection of reference genes for RT-qPCRanalysis in the monarch butterfly,Danaus plexippus(L.),a migrating bio-Indicator.PLoS One,2015,10:e0129482.
[17]Liu G Q,Qiu X H,Cao L,Zhang Y,Zhan Z B,Han R C.Evaluation of reference genes for reverse transcription quantitative PCR studies of physiological responses in the ghost moth,Thitarodes armoricanus(Lepidoptera,Hepialidae).PLoS One,2016,11:e0159060.
[18]Zhang L,Zhang Q L,Wang X T,Yang X Z,Li X P,Yuan M L.Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars(Lepidoptera:Erebidae:Gynaephora)along an altitude gradient.Ecol Evol,2017,7:9054-9065.
[19]杨苓,胡晓静,徐志峰,何林,肖伟.桃蛀螟实时荧光定量PCR内参基因的筛选.昆虫学报,2017,60(11):1266-1277.Yang L,Hu X J,Xu Z F,He L,Xiao W.Screening of reference genes for qRT-PCR in Conogethes punctiferails(Lepidoptera:Crambidae).Acta Entomol Sin,2017,60(11):1266-1277.(in Chinese with English abstract)
[20]Teng X L,Zhang Z,He G L,Yang L W,Li F.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.J Insect Sci,2011,12:60.
[21]Xu J,Lu M X,Cui Y D,Du Y Z.Selection and evaluation of reference genes for expression analysis using qRT-PCR in Chilo suppressalis(Lepidoptera:Pyralidae).J Econ Entomol,2017,110:683-691.
[22]胡阳,郑永利,曹国连,傅强.利用半人工饲料大规模简便化饲养二化螟中国水稻科学,2013,27(5):535-538.Hu Y,Zheng Y L,Cao G L,Fu Q.A technique for rearing Chilo suppressalis in the large scale with an oligidic diet in laboratory.Chin J Rice Sci,2013,27(5):535-538.(in Chinese with English abstract)
[23]吴敏,张真真,高聪芬.水稻二化螟抗药性监测方法.应用昆虫学报,2013,50(2):548-552.Wu M,Zhang Z Z,Gao C F.Methods of insecticides resistance monitoring of the striped stem borer,Chilo suppressalis.Chin J Appl Entomol,2013,50(2):548-552.(in Chinese with English abstract)
[24]Hui X M,Yang W,He G L,Yang Q P,Han Z,Li F.RNAinterference of ace1 and ace2 in Chilo suppressalis reveals their different contributions to motor ability and larval growth.Insect Mol Biol,2011,4:507-518.
[25]Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity.BestKeeper:Excel-based tool using pair-wise correlations.Biotechnol Lett,2004,26:509-515.
[26]Andersen C L,Jensen J L,Orntoft T F.Normalization of real-time quantitative reverse transcription-PCR data:Amodel-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Res,2004,64:5245-5250.
[27]Schmittgen T D,Livak K J.Analyzing real-time PCRdata by the comparative CT method.Nat Protoc,2008,3:1101-1108.
[28]Radonic A,Thulke S,Mackay I M,Landt O,Siegert W,Nitsche A.Guideline to reference gene selection for quantitative real-time PCR.Biochem Biophys Res Commun,2004,313:856-862.
[29]Huis R,Hawkins S,Neutelings G.Selection of reference genes for quantitative gene expression normalization in flax(Linum usitatissimum L.).BMC Plant Biol,2010,10:71.
[30]Kozera B,Rapacz M.Reference genes in real-time PCR.J Appl Genet,2013,54:391-406.
[31]冯波,郭前爽,毛必鹏,杜永均.松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选.昆虫学报,2016,59(4):427-437.Feng B,Guo Q,Mao B P,Du Y J.Identification and selection of valid reference genes for assaying gene expression in the chemosensory tissues of Monochamus alternatus(Coleoptera:Cerambycidae)by RT-PCR.Acta Entomol Sin,2016,59(4):427-437.(in Chinese with English abstract)
[32]Nielsen M G,Gadagkar S R,Gutzwiller L.Tubulin evolution in insects:Gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family.BMC Evol Biol,2010,10:113.
[33]Yuan M,Lu Y H,Zhu X,Wan H,Shakeel M,Zhan S,Jin B R,Li J H.Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)using reverse-transcription quantitative PCR.PLoS One,2014,9:e86503.
[34]An X K,Hou M L,Liu Y D.Reference gene selection and evaluation for gene expression studies using qRT-PCR in the white-backed planthopper,Sogatella furcifera(Hemiptera:Delphacidae).J Econ Entomol,2016,109:879-886.
[35]Dai T M,Lu Z C,Liu W X,Wan F H.Selection and validation of reference genes for qRT-PCR analysis during biological invasions:The thermal adaptability of Bemisia tabaci MED.PLoS One,2017,12:e0173821.
[36]Ponton F,Chapuis M P,Pernice M,Sword GA,Simpson S J.Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster.J Insect Physiol,2011,57:840-850.
[37]Yang Q P,Li Z,Cao J J,Zhang S D,Zhang H J,Wu X Y,Zhang Q W,Liu X X.Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria(Orthoptera:Acrididae).PLoS One,2014,9:e98164.
[38]Zheng Y T,Li H B,Lu M X,Du Y Z.Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis(Thysanoptera:Thripidae).PLoS One,2014,9:e111369.
[39]Pan H P,Yang X W,Siegfried B D,Zhou X G.Acomprehensive selection of reference genes for RT-qPCRanalysis in a predatory lady beetle,Hippodamia convergens(Coleoptera:Coccinellidae).PLoS One,2015,10:e0125868.
[40]Qiu L,Wang P,Wu T,Li B,Wang X,Lei C,Lin Y,Zhao J,Ma W.Downregulation of Chilo suppressalis alkaline phosphatase genes associated with resistance to three transgenic Bacillus thuringiensis rice lines.Insect Mol Biol,2018,27:83-89.
[41]Xu G,Gu G X,Teng Z W,Wu S F,Huang J,Song Q S,Ye G Y,Fang Q.Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.Sci Rep,2016,6:28976.
[42]Xu G,Wu S F,Teng Z W,Yao H W,Fang Q,Huang J,Ye G Y.Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer,Chilo suppressalis(Lepidoptera:Crambidae).Insect Sci,2017,24:371-384.
[43]Xu G,Wu S F,Wu Y S,Gu G X,Fang Q,Ye G Y.De novo assembly and characterization of central nervous system transcriptome reveals neurotransmitter signaling systems in the rice striped stem borer,Chilo suppressalis.BMC Genomics,2015,16:525.