Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis(Hemiptera: Pseudococcidae) sex-dimorphic development
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摘要
Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.
Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.
Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.
引文
Andersen C L,Jensen J L,?rntoft T F.2004.Normalization of real-time quantitative reverse transcription-PCR data:A model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Research,64,5245.
Arya S K,Jain G,Upadhyay S K,Sarita,Singh H,Dixit S,Verma P C.2017.Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.Scientific Reports,7,13520.
Brent C S,Hull J J.2014.Characterization of male-derived factors inhibiting female sexual receptivity in Lygus hesperus.Journal of Insect Physiology,60,104-110.
Brent C S,Miyasaki K,Vuong C,Miranda B,Steele B,Brent K G,Nath R.2016.Regulatory roles of biogenic amines and juvenile hormone in the reproductive behavior of the western tarnished plant bug(Lygus hesperus).Journal of Comparative Physiology(B:Biochemical Systemic&Environmental Physiology),186,169-179.
Bustin S A.2002.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):Trends and problems.Journal of Molecular Endocrinology,29,23-39.
Bustin S A,Benes V,Garson J A,Hellemans J,Huggett J,Kubista M,Mueller R,Nolan T,Pfaffl M W,Shipley G L,Vandesompele J,Wittwer C T.2009.The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments.Clinical Chemistry,55,611-622.
Chapuis M P,Tohidiesfahani D,Dodgson T.2011.Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust.BMCMolecular Biology,12,1-11.
Chen F,Lu Y Y.2014.Selection of reference genes in Phenacoccus solenopsis(Hemiptera:Pseudococcidae)under heat stress.Acta Entomologica Sinica,57,1146-1154.(in Chinese)
Fu W,Xie W,Zhang Z,Wang S,Wu Q,Liu Y,Zhou X,Zhou X,Zhang Y.2013.Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella(Lepidoptera:Plutellidae).International Journal of Biological Sciences,9,792-802.
Godfrey N,Simon H,Rudy H.2010.Selection of reference genes for quantitative gene expression normalization in flax(Linum usitatissimum L.).BMC Plant Biology,10,1-14.
Gullan P J,Kosztarab M.1997.Adaptations in scale insects.Annual Review of Entomology,42,23.
Guo J L,Ling H,Wu Q B,Que Y X.2014.The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.Scientific Reports,4,7042.
Gutierrez L,Mauriat M,Guénin S,Pelloux J,Lefebvre JF,Louvet R,Rusterucci C,Moritz T,Guerineau F,Bellini C,Van Wuytswinkel O.2008.The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR)analysis in plants.Plant Biotechnology Journal,6,609.
Heid C A,Stevens J,Livak K J,Williams P M.1996.Real time quantitative PCR.Genome Research,6,986-994.
Van Hiel M B V,Wielendaele P V,Temmerman L,Van Soest S,Vuerinckx K,Huybrechts R,Broeck J V,Simonet G.2009.Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions.BMC Molecular Biology,10,56.
Hoogewijs D,Houthoofd K,Matthijssens F,Vandesompele J,Vanfleteren J R.2008.Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C.elegans.BMC Molecular Biology,9,9.
Hu J J,Meng X,Zhou J B,Yang L X,Liu S H,Li R Z.2017.Transcriptome analysis of the cotton mealybug,Phenacoccus solenopsis(Hemiptera:Pseudococcidae).Acta Entomologica Sinica,60,9-17.(in Chinese)
Jacob F,Guertler R,Naim S,Nixdorf S,Fedier A,Hacker NF,Heinzelmann-Schwarz V.2013.Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.PLoSONE,8,e59180.
Jiang H B,Liu Y H,Tang P A,Zhou A W,Wang J J.2010.Validation of endogenous reference genes for insecticideinduced and developmental expression profiling of Liposcelis bostsrychophila(Psocoptera:Liposcelididae).Molecular Biology Reports,37,1019-1029.
Langnaese K,John R,Schweizer H,Ebmeyer U,Keilhoff G.2008.Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model.BMC Molecular Biology,9,53.
Li R M,Xie W,Wang S L,Wu Q J,Yang N,Yang X,Pan H P,Zhou X M,Bai L Y,Xu B Y,Zhou X G,Zhang Y J.2013.Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly,Bemisia tabaci(Hemiptera:Aleyrodidae).PLoS ONE,8,e53006.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-??CT method.Methods,25,402-408.
Li W,Huang Z Y,Liu F,Li Z,Yan L,Zhang S,Chen S,Zhong B,Su S.2013.Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee,Apis mellifera,and its differential expression during caste differentiation.PLoS ONE,8,e68544.
Liu G Q,Qiu X H,Cao L,Zhang Y,Zhan Z B,Han R C.2016.Evaluation of reference genes for reverse transcription quantitative pcr studies of physiological responses in the ghost moth,Thitarodes armoricanus(Lepidoptera,Hepialidae).PLoS ONE,11,e0159060.
Mamidala P,Rajarapu S P,Jones S C,Mittapalli O.2011.Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.Journal of Medical Entomology,48,947-951.
Mao J J,Zeng F R.2014.Plant-mediated RNAi of a gap geneenhanced tobacco tolerance against the Myzus persicae.Transgenic Research,23,145-152.
Marchal E,Zhang J R,Badisco L,Verlinden H,Hult E F,Van Wielendaele P,Yagi K J,Tobe S S,Vanden Broeck J.2011.Final steps in juvenile hormone biosynthesis in the desert locust,Schistocerca gregaria.Insect Biochemistry&Molecular Biology,41,219-227.
Minakuchi C,Namiki T,Yoshiyama M,Shinoda T.2008.RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum.FEBS Journal,275,2919-2931.
Niu J,Cappelle K,de Miranda J R,Smagghe G,Meeus I.2014.Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris.Journal of Invertebrate Pathology,115,76.
Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeperExcel-based tool using pair-wise correlations.Biotechnology Letters,26,509-515.
Ponton F,Chapuis M P,Pernice M,Sword G A,Simpson S J.2011.Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster.Journal of Insect Physiology,57,840-850.
Pszczolkowski M A,Tucker A,Srinivasan A,Ramaswamy SB.2006.On the functional significance of juvenile hormone in the accessory sex glands of male Heliothis virescens.Journal of Insect Physiology,52,786-794.
Ross D T,Scherf U,Eisen M B,Perou C M,Rees C,Spellman P,Iyer V,Jeffrey S S,Van de Rijn M,Waltham M,Pergamenschikov A,Lee J C,Lashkari D,Shalon D,Myers T G,Weinstein J N,Botstein D,Brown P O.2000.Systematic variation in gene expression patterns in human cancer cell lines.Nature Genetics,24,227-235.
Scharlaken B,Graaf D C D,Goossens K,Brunain M,Peelman L J,Jacobs F J.2008.Reference gene selection for insect expression studies using quantitative real-time PCR:The Head of the honeybee,Apis mellifera,after a bacterial challenge.Journal of Insect Science,8,1-10.
Shen G M,Jiang H B,Wang X N,Wang J J.2010.Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis(Diptera:Tephritidae).BMC Molecular Biology,11,76.
Shi C H,Yang F S,Zhu X,Du E X,Yang Y T,Wang S L,Wu QJ,Zhang Y J.2016.Evaluation of Housekeeping genes for quantitative real-time PCR analysis of Bradysia odoriphaga(Diptera:Sciaridae).International Journal of Molecular Sciences,17,1034.
Shi X Q,Guo W C,Wan P J,Zhou L T,Ren X L,Ahmat T,Fu K Y,Li G Q.2013.Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata(Say).BMC Research Notes,6,93.
Shinoda T,Itoyama K.2003.Juvenile hormone acid methyltransferase:A key regulatory enzyme for insect metamorphosis.Proceedings of the National Academy of Sciences of the United States of America,100,11986-11991.
Shirk P D,Bhaskaran G,R?ller H.1980.The transfer of juvenile hormone from male to female during mating in the Cecropia silkmoth.Experientia,36,682-683.
Silver N,Best S,Jiang J,Thein S L 2006.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.BMC Molecular Biology,7,1-9.
Sinha D K,Smith C M.2014.Selection of reference genes for expression analysis in Diuraphis noxia(Hemiptera:Aphididae)fed on resistant and susceptible wheat plants.Scientific Reports,4,5059.
Sun M,Lu M X,Tang X T,Du Y Z.2015.Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens(Lepidoptera:Noctuidae).PLoS ONE,10,e0115979.
Teng X L,Zhang Z,He G L,Yang L,Li F.2012.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.Journal of Insect Science,12,1-17.
Vandesompele J,Paepe A D,Speleman F.2002a.Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR.Analytical Biochemistry,303,95-98.
Vandesompele J,Preter K D,Pattyn F,Poppe B,Roy N V,Paepe A D,Speleman F.2002b.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biology,3,1-11.
Vea I M,Tanaka S,Shiotsuki T,Jouraku A,Tanaka T,Minakuchi C.2016.Differential juvenile hormone variations in scale insect extreme sexual dimorphism.PLoS ONE,11,e0149459.
Wan P J,Tang Y H,Yuan S Y,He J C,Wang W X,Lai F X,Fu Q.2017.Reference genes for quantitative real-time PCR analysis in symbiont entomomyces delphacidicola of Nilaparvata lugens(St?l).Scientific Reports,7,42206.
Willems E,Mateizel I,Kemp C,Cauffman G,Sermon K,Leyns L.2006.Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.International Journal of Developmental Biology,50,627.
Nie X P,Li Q L,Xu C,Li D Z,Zhang Z,Wang M Q,Zhou AM,Wang M Q.2018.Antennal transcriptome and odorant binding protein expression profiles of an invasive mealybug and its parasitoid.Journal of Applied Entomology,142,149-161.
Yang C X,Li H,Pan H P,Ma Y,Zhang D,Liu Y,Zhang Z,Zheng C,Chu D.2015.Stable reference gene selection for RT-qPCR analysis in nonviruliferous and viruliferous Frankliniella occidentalis.PLoS ONE,10,e0135207.
Yang C X,Pan H P,Liu Y,Zhou X.2014.Selection of reference genes for expression analysis using quantitative realtime PCR in the pea aphid,Acyrthosiphon pisum(Harris)(Hemiptera,Aphidiae).PLoS ONE,9,e110454.
Yuan M,Lu Y H,Zhu X,Wan H,Shakeel M,Zhan S,Jin B R,Li J.2014.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,9,e86503.
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.2015.Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae).Gene,555,393-402.
Zhang Z J,Zhang P J,Li W D,Zhang J M,Huang F,Yang J,Bei Y W,Lu Y B.2013.De novo transcriptome sequencing in Frankliniella occidentalis,to identify genes involved in plant virus transmission and insecticide resistance.Genomics,101,296-305.
Zheng Y T,Li H B,Lu M X,Du Y Z.2014.Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis(Thysanoptera:Thripidae).PLo SONE,9,e111369.
Arya S K,Jain G,Upadhyay S K,Sarita,Singh H,Dixit S,Verma P C.2017.Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.Scientific Reports,7,13520.
Brent C S,Hull J J.2014.Characterization of male-derived factors inhibiting female sexual receptivity in Lygus hesperus.Journal of Insect Physiology,60,104-110.
Brent C S,Miyasaki K,Vuong C,Miranda B,Steele B,Brent K G,Nath R.2016.Regulatory roles of biogenic amines and juvenile hormone in the reproductive behavior of the western tarnished plant bug(Lygus hesperus).Journal of Comparative Physiology(B:Biochemical Systemic&Environmental Physiology),186,169-179.
Bustin S A.2002.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):Trends and problems.Journal of Molecular Endocrinology,29,23-39.
Bustin S A,Benes V,Garson J A,Hellemans J,Huggett J,Kubista M,Mueller R,Nolan T,Pfaffl M W,Shipley G L,Vandesompele J,Wittwer C T.2009.The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments.Clinical Chemistry,55,611-622.
Chapuis M P,Tohidiesfahani D,Dodgson T.2011.Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust.BMCMolecular Biology,12,1-11.
Chen F,Lu Y Y.2014.Selection of reference genes in Phenacoccus solenopsis(Hemiptera:Pseudococcidae)under heat stress.Acta Entomologica Sinica,57,1146-1154.(in Chinese)
Fu W,Xie W,Zhang Z,Wang S,Wu Q,Liu Y,Zhou X,Zhou X,Zhang Y.2013.Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella(Lepidoptera:Plutellidae).International Journal of Biological Sciences,9,792-802.
Godfrey N,Simon H,Rudy H.2010.Selection of reference genes for quantitative gene expression normalization in flax(Linum usitatissimum L.).BMC Plant Biology,10,1-14.
Gullan P J,Kosztarab M.1997.Adaptations in scale insects.Annual Review of Entomology,42,23.
Guo J L,Ling H,Wu Q B,Que Y X.2014.The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.Scientific Reports,4,7042.
Gutierrez L,Mauriat M,Guénin S,Pelloux J,Lefebvre JF,Louvet R,Rusterucci C,Moritz T,Guerineau F,Bellini C,Van Wuytswinkel O.2008.The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR)analysis in plants.Plant Biotechnology Journal,6,609.
Heid C A,Stevens J,Livak K J,Williams P M.1996.Real time quantitative PCR.Genome Research,6,986-994.
Van Hiel M B V,Wielendaele P V,Temmerman L,Van Soest S,Vuerinckx K,Huybrechts R,Broeck J V,Simonet G.2009.Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions.BMC Molecular Biology,10,56.
Hoogewijs D,Houthoofd K,Matthijssens F,Vandesompele J,Vanfleteren J R.2008.Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C.elegans.BMC Molecular Biology,9,9.
Hu J J,Meng X,Zhou J B,Yang L X,Liu S H,Li R Z.2017.Transcriptome analysis of the cotton mealybug,Phenacoccus solenopsis(Hemiptera:Pseudococcidae).Acta Entomologica Sinica,60,9-17.(in Chinese)
Jacob F,Guertler R,Naim S,Nixdorf S,Fedier A,Hacker NF,Heinzelmann-Schwarz V.2013.Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.PLoSONE,8,e59180.
Jiang H B,Liu Y H,Tang P A,Zhou A W,Wang J J.2010.Validation of endogenous reference genes for insecticideinduced and developmental expression profiling of Liposcelis bostsrychophila(Psocoptera:Liposcelididae).Molecular Biology Reports,37,1019-1029.
Langnaese K,John R,Schweizer H,Ebmeyer U,Keilhoff G.2008.Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model.BMC Molecular Biology,9,53.
Li R M,Xie W,Wang S L,Wu Q J,Yang N,Yang X,Pan H P,Zhou X M,Bai L Y,Xu B Y,Zhou X G,Zhang Y J.2013.Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly,Bemisia tabaci(Hemiptera:Aleyrodidae).PLoS ONE,8,e53006.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-??CT method.Methods,25,402-408.
Li W,Huang Z Y,Liu F,Li Z,Yan L,Zhang S,Chen S,Zhong B,Su S.2013.Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee,Apis mellifera,and its differential expression during caste differentiation.PLoS ONE,8,e68544.
Liu G Q,Qiu X H,Cao L,Zhang Y,Zhan Z B,Han R C.2016.Evaluation of reference genes for reverse transcription quantitative pcr studies of physiological responses in the ghost moth,Thitarodes armoricanus(Lepidoptera,Hepialidae).PLoS ONE,11,e0159060.
Mamidala P,Rajarapu S P,Jones S C,Mittapalli O.2011.Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius.Journal of Medical Entomology,48,947-951.
Mao J J,Zeng F R.2014.Plant-mediated RNAi of a gap geneenhanced tobacco tolerance against the Myzus persicae.Transgenic Research,23,145-152.
Marchal E,Zhang J R,Badisco L,Verlinden H,Hult E F,Van Wielendaele P,Yagi K J,Tobe S S,Vanden Broeck J.2011.Final steps in juvenile hormone biosynthesis in the desert locust,Schistocerca gregaria.Insect Biochemistry&Molecular Biology,41,219-227.
Minakuchi C,Namiki T,Yoshiyama M,Shinoda T.2008.RNAi-mediated knockdown of juvenile hormone acid O-methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum.FEBS Journal,275,2919-2931.
Niu J,Cappelle K,de Miranda J R,Smagghe G,Meeus I.2014.Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris.Journal of Invertebrate Pathology,115,76.
Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeperExcel-based tool using pair-wise correlations.Biotechnology Letters,26,509-515.
Ponton F,Chapuis M P,Pernice M,Sword G A,Simpson S J.2011.Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster.Journal of Insect Physiology,57,840-850.
Pszczolkowski M A,Tucker A,Srinivasan A,Ramaswamy SB.2006.On the functional significance of juvenile hormone in the accessory sex glands of male Heliothis virescens.Journal of Insect Physiology,52,786-794.
Ross D T,Scherf U,Eisen M B,Perou C M,Rees C,Spellman P,Iyer V,Jeffrey S S,Van de Rijn M,Waltham M,Pergamenschikov A,Lee J C,Lashkari D,Shalon D,Myers T G,Weinstein J N,Botstein D,Brown P O.2000.Systematic variation in gene expression patterns in human cancer cell lines.Nature Genetics,24,227-235.
Scharlaken B,Graaf D C D,Goossens K,Brunain M,Peelman L J,Jacobs F J.2008.Reference gene selection for insect expression studies using quantitative real-time PCR:The Head of the honeybee,Apis mellifera,after a bacterial challenge.Journal of Insect Science,8,1-10.
Shen G M,Jiang H B,Wang X N,Wang J J.2010.Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis(Diptera:Tephritidae).BMC Molecular Biology,11,76.
Shi C H,Yang F S,Zhu X,Du E X,Yang Y T,Wang S L,Wu QJ,Zhang Y J.2016.Evaluation of Housekeeping genes for quantitative real-time PCR analysis of Bradysia odoriphaga(Diptera:Sciaridae).International Journal of Molecular Sciences,17,1034.
Shi X Q,Guo W C,Wan P J,Zhou L T,Ren X L,Ahmat T,Fu K Y,Li G Q.2013.Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata(Say).BMC Research Notes,6,93.
Shinoda T,Itoyama K.2003.Juvenile hormone acid methyltransferase:A key regulatory enzyme for insect metamorphosis.Proceedings of the National Academy of Sciences of the United States of America,100,11986-11991.
Shirk P D,Bhaskaran G,R?ller H.1980.The transfer of juvenile hormone from male to female during mating in the Cecropia silkmoth.Experientia,36,682-683.
Silver N,Best S,Jiang J,Thein S L 2006.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.BMC Molecular Biology,7,1-9.
Sinha D K,Smith C M.2014.Selection of reference genes for expression analysis in Diuraphis noxia(Hemiptera:Aphididae)fed on resistant and susceptible wheat plants.Scientific Reports,4,5059.
Sun M,Lu M X,Tang X T,Du Y Z.2015.Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens(Lepidoptera:Noctuidae).PLoS ONE,10,e0115979.
Teng X L,Zhang Z,He G L,Yang L,Li F.2012.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.Journal of Insect Science,12,1-17.
Vandesompele J,Paepe A D,Speleman F.2002a.Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR.Analytical Biochemistry,303,95-98.
Vandesompele J,Preter K D,Pattyn F,Poppe B,Roy N V,Paepe A D,Speleman F.2002b.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biology,3,1-11.
Vea I M,Tanaka S,Shiotsuki T,Jouraku A,Tanaka T,Minakuchi C.2016.Differential juvenile hormone variations in scale insect extreme sexual dimorphism.PLoS ONE,11,e0149459.
Wan P J,Tang Y H,Yuan S Y,He J C,Wang W X,Lai F X,Fu Q.2017.Reference genes for quantitative real-time PCR analysis in symbiont entomomyces delphacidicola of Nilaparvata lugens(St?l).Scientific Reports,7,42206.
Willems E,Mateizel I,Kemp C,Cauffman G,Sermon K,Leyns L.2006.Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.International Journal of Developmental Biology,50,627.
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