温度和药剂胁迫下茶刺盲蝽内参基因稳定性分析
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摘要
为筛选特定条件下实时荧光定量PCR检测体系中能稳定表达的茶刺盲蝽Helopeltis theivora内参基因,从其转录组中筛选并克隆11个候选基因Actin、β-tubulin1、RPL13A、EF1α、RPS3A、GAPDH、18S RNA、G6PDH、EIF4A、TBP和UBQ,测定它们在不同温度及药剂胁迫下mRNA的表达水平,并通过geNorm、NormFinder、BestKeeper、Delta Ct和RefFinder软件(算法)分析各基因的稳定性。结果表明,11个候选基因引物均具有良好的特异性和扩增效率(90.10%～96.87%)。温度胁迫下,EIF4A有最小的平均变异度(mean variability,M)、稳定值(stability value,SV)和几何平均值,分别为0.200、0.096和2.060;RPS3A的平均标准偏差(average of standard deviation,STDEV)最小,为0.605;二者是表达最稳定的基因。药剂胁迫下,RPL13A的M值和STDEV最小,分别为0.123和0.660;RPS3A的SV、标准偏差(standard deviation,SD)和几何平均值均最小,分别为0.063、0.336和1.189;二者是表达最稳定的基因;Actin为最不稳定基因。全样品评价中,RPL13A(M值为0.282、几何平均值为1.565)和RPS3A(SV为0.099、STDEV为0.614)的评价值同比最小,是表达最稳定的基因;最不稳定的基因为Actin、β-tubulin1和TBP。geNorm分析结果还显示所有处理条件下最适内参基因数目均为2个。表明EIF4A和RPS3A可作为茶刺盲蝽与温度相关的抗性基因表达研究的内参基因,RPS3A和RPL13A可作为该虫抗药基因表达研究的内参基因。
In order to select suitable reference genes for quantitative real-time PCR(qPCR) in tea mosquito bug Helopeltis theivora, 11 candidate reference genes, including Actin, β-tubulin1, RPL13A,EF1α, RPS3A, GAPDH, 18S RNA, G6PDH, EIF4A, TBP and UBQ, were identified and cloned based on the transcriptome data of H. theivora. The mRNA expression levels of these candidate genes under different temperature and pesticide stresses were measured by qPCR, and their expression stability was evaluated respectively by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms. The results showed that the primers of the 11 candidate genes showed high amplification efficiency and specificity(90.10%-96.87%). Under temperature stress, EIF4A with the lowest mean variability(M) value(0.200), stability value(SV)(0.096) and geomean(2.060), and RPS3A with the minimum average of standard deviation(STDEV)(0.605), were recommended as the most stable genes. Under pesticide stress, RPL13A with the lowest M value(0.123) and STDEV(0.660), and RPS3A with the lowest SV(0.063), standard deviation(SD) value(0.336) and geomean(1.189), were ranked as the most stable genes. However, Actin was confirmed as the least stable gene by all algorithms. In all samples, RPL13A(with minimum M value of 0.282 and geomean of 1.565) and RPS3A(with minimum SV of 0.099 and STDEV of 0.614) were both considered as the most stable genes. Actin, β-tubulin1 and TBP were ranked as the least stable genes. Additionally, two optimal reference genes were recommended by geNorm among different treatments. The results indicated that EIF4A and RPS3A could be used as reference genes for study on resistance gene expression under temperature stress in H. theivora, while RPS3A and RPL13A could be used as reference genes for expression analysis under pesticide stress.
In order to select suitable reference genes for quantitative real-time PCR(qPCR) in tea mosquito bug Helopeltis theivora, 11 candidate reference genes, including Actin, β-tubulin1, RPL13A,EF1α, RPS3A, GAPDH, 18S RNA, G6PDH, EIF4A, TBP and UBQ, were identified and cloned based on the transcriptome data of H. theivora. The mRNA expression levels of these candidate genes under different temperature and pesticide stresses were measured by qPCR, and their expression stability was evaluated respectively by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms. The results showed that the primers of the 11 candidate genes showed high amplification efficiency and specificity(90.10%-96.87%). Under temperature stress, EIF4A with the lowest mean variability(M) value(0.200), stability value(SV)(0.096) and geomean(2.060), and RPS3A with the minimum average of standard deviation(STDEV)(0.605), were recommended as the most stable genes. Under pesticide stress, RPL13A with the lowest M value(0.123) and STDEV(0.660), and RPS3A with the lowest SV(0.063), standard deviation(SD) value(0.336) and geomean(1.189), were ranked as the most stable genes. However, Actin was confirmed as the least stable gene by all algorithms. In all samples, RPL13A(with minimum M value of 0.282 and geomean of 1.565) and RPS3A(with minimum SV of 0.099 and STDEV of 0.614) were both considered as the most stable genes. Actin, β-tubulin1 and TBP were ranked as the least stable genes. Additionally, two optimal reference genes were recommended by geNorm among different treatments. The results indicated that EIF4A and RPS3A could be used as reference genes for study on resistance gene expression under temperature stress in H. theivora, while RPS3A and RPL13A could be used as reference genes for expression analysis under pesticide stress.
引文
Andersen CL,Jensen JL,?rntoft TF.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(15):5245-5250
Arya SK,Jain G,Upadhyay SK,Sarita,Singh H,Dixit S,Verma PC.2017.Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.Scientific Reports,7(1):13520
Bansal R,Mamidala P,Mian MA,Mittapalli O,Michel AP.2012.Validation of reference genes for gene expression studies in Aphis glycines(Hemiptera:Aphididae).Journal of Economic Entomology,105(4):1432-1438
Bustin SA,Benes V,Garson JA,Hellemans J,Huggett J,Kubista M,Mueller R,Nolan T,Pfaffl MW,Shipley GL,et al.2009.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments.Clinical Chemistry,55(4):611-622
Chandrashekara KN,Kumar RR,Kumar V,Banerjee D,Kundu S,Ghosh B,Tyagi K.2015.DNA barcoding reveals host associated genetic diversity of‘tea mosquito bug’Helopeltis theivora(Miridae:Heteroptera)from India.Journal of Asia-Pacific Entomology,18(3):541-545
Chen F,Lu YY.2014.Selection of reference genes in Phenacoccus solenopsis(Hemiptera:Pseudococcidae)under heat stress.Acta Entomologica Sinica,57(10):1146-1154(in Chinese)[陈芳,陆永跃.2014.热胁迫下棉花粉蚧内参基因的筛选.昆虫学报,57(10):1146-1154]
Chen H,Yang ZQ,Hu Y,Tan JH,Jia J,Xu HL,Chen XH.2016.Reference genes selection for quantitative gene expression studies in Pinus massoniana L.Trees,30(3):685-696
Concha C,Edman RM,Belikoff EJ,Schiemann AH,Carey B,Scott MJ.2012.Organization and expression of the Australian sheep blowfly(Lucilia cuprina)hsp23,hsp24,hsp70 and hsp83 genes.Insect Molecular Biology,21(2):169-180
Dai TM,LüZC,Liu WX,Wan FH.2017.Selection and validation of reference genes for qRT-PCR analysis during biological invasions:the thermal adaptability of Bemisia tabaci MED.PLoSONE,12(3):e0173821
Dzaki N,Ramli KN,Azlan A,Ishak IH,Azzam G.2017.Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.Scientific Reports,7:43618
Ibanez F,Tamborindeguy C.2016.Selection of reference genes for expression analysis in the potato psyllid,Bactericera cockerelli.Insect Molecular Biology,25(3):227-238
Jian B,Liu B,Bi YR,Hou WS,Wu CX,Han TF.2008.Validation of internal control for gene expression study in soybean by quantitative real-time PCR.BMC Molecular Biology,9:59
Koramutla MK,Aminedi R,Bhattacharya R.2016.Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid,Lipaphis erysimi(Kalt).Scientific Reports,6:25883
Ladror DT,Frey BL,Scalf M,Levenstein ME,Artymiuk JM,Smith LM.2014.Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions.Biochemical and Biophysical Research Communications,445(3):535-541
Liang P,Guo Y,Zhou X,Gao X.2014.Expression profiling in Bemisia tabaci under insecticide treatment:indicating the necessity for custom reference gene selection.PLoS ONE,9(1):e87514
Liu GQ,Qiu XH,Cao L,Zhan ZB,Han RC.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(7):e0159060
Lu Y,Yuan M,Gao X,Kang T,Zhan S,Wan H,Li J.2013.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLoS ONE,8(7):e68059
Luo YM,Jin QA.1991.A study on the mosquito bug infesting cashew in Hainan Island.Acta Entomologica Sinica,34(1):60-67(in Chinese)[罗永明,金启安.1991.海南岛腰果角盲蝽的研究.昆虫学报,34(1):60-67]
Nagy NA,Németh Z,Juhász E,Póliska S,Rácz R,Kosztolányi A,Barta Z.2017.Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle,Lethrus apterus(Coleoptera:Geotrupidae).PeerJ,5:e4047
Pan HP,Yang XW,Bidne K,Hellmich RL,Siegfried BD,Zhou XG,Zhang YJ.2015.Selection of reference genes for RT-qPCR analysis in the monarch butterfly,Danaus plexippus(L.),a migrating bio-indicator.PLoS ONE,10(6):e0129482
Pfaffl MW,Tichopad A,Prgomet C,Neuvians TP.2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations.Biotechnology Letters,26(6):509-515
P?achetka-Bo?ek A,Augustyniak M.2017.Evaluation of candidate reference genes for quantitative gene expression analysis in Spodoptera exigua after long-time exposure to cadmium.Scientific Reports,7(1):8338
Ponton F,Chapuis MP,Pernice M,Sword GA,Simpson SJ.2011.Evaluation of potential reference genes for reverse transcription-qP-CR studies of physiological responses in Drosophila melanogaster.Journal of Insect Physiology,57(6):840-850
Roy S,Muraleedharan N,Mukhapadhyay A,Handique G.2015.The tea mosquito bug,Helopeltis theivora Waterhouse(Heteroptera:Miridae):its status,biology,ecology and management in tea plantations.International Journal of Pest Management,61(3):179-197
Scharlaken B,de Graaf DC,Goossens K,Brunain M,Peelman LJ,Jacobs FJ.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,8:33
Shen GM,Jiang HB,Wang XN,Wang JJ.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
Shu BS,Zhang JJ,Cui GF,Sun RR,Sethuraman V,Yi X,Zhong GH.2018.Evaluation of reference genes for real-time quantitative PCR analysis in larvae of Spodoptera litura exposed to azadirachtin stress conditions.Frontiers in Physiology,9:372
Silver N,Best S,Jiang J,Thein SL.2006.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.BMC Molecular Biology,7:33
Simmons J,D’Souza O,Rheault M,Donly C.2013.Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.Insect Molecular Biology,22(1):62-71
Srikumar KK,Bhat PS.2013.Biology of the tea mosquito bug(Helopeltis theivora Waterhouse)on Chromolaena odorata(L.)R.M.King&H.Rob.Chilean Journal of Agricultural Research,73(3):309-314
Su L,Meng JY,Zhu JM,Zhang CY.2018.Cloning and expression of Hsp90 gene from green peach aphid Myzus persicae under UV-Bstress.Journal of Plant Protection,45(6):1267-1273(in Chinese)[苏丽,孟建玉,朱佳敏,张长禹,2018.烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析.植物保护学报,45(6):1267-1273]
Svitkin YV,Pause A,Haghighat A,Pyronnet S,Witherell G,Belsham GJ,Sonenberg N.2001.The requirement for eukaryotic initiation factor 4A(eIF4A)in translation is in direct proportion to the degree of mRNA 5’secondary structure.RNA,7(3):382-394
Taylor S,Wakem M,Dijkman G,Alsarraj M,Nguyen M.2010.A practical approach to RT-qPCR-publishing data that conform to the MIQE guidelines.Methods,50(S):1-5
Teng XL,Zhang Z,He GL,Yang LW,Li F.2012.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.Journal of Insect Science,12:60
Vandesompele J,de Preter K,Pattyn F,Poppe B,van Roy N,de Paepe A,Speleman F.2002.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biology,3(7):research0034
Volkov RA,Panchuk II,Sch?ffl F.2003.Heat-stress-dependency and developmental modulation of gene expression:the potential of house-keeping genes as internal standards in m RNA expression profiling using real-time RT-PCR.Journal of Experimental Botany,54(391):2343-2349
Wang B,Du HH,Yao ZP,Ren C,Ma L,Wang J,Zhang H,Ma H.2018.Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses.Physiology and Molecular Biology of Plant,24(3):455-463
Wang Z,Meng QQ,Liu AQ,Sang LW,Sun SW,Gou YF,Gao SF.2017.Observation of the sensilla on antenna and propodium of Helopeltis theivora Waterhouse(Hemiptera:Mirada)with scanning electron microscope.Chinese Journal of Tropical Crops,38(11):2165-2170(in Chinese)[王政,孟倩倩,刘爱勤,桑利伟,孙世伟,苟亚峰,高圣风.2017.茶角盲蝽触角和前足感器的扫描电镜观察.热带作物学报,38(11):2165-2170]
Wu S,Zhang XF,He YQ,Shuai JB,Chen XM,Ling EJ.2010.Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development.Developmental&Comparative Immunology,34(11):1191-1198
Xie FL,Sun GL,Stiller JW,Zhang BH.2011.Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database.PLoS ONE,6(11):e26980
Yang L,Hu XJ,Xu ZF,He L,Xiao W.2017.Screening of reference genes for qRT-PCR in Conogethes punctiferalis(Lepidoptera:Crambidae).Acta Entomologica Sinica,60(11):1266-1277(in Chinese)[杨苓,胡晓静,徐志峰,何林,肖伟.2017.桃蛀螟实时荧光定量PCR内参基因的筛选.昆虫学报,60(11):1266-1277]
Yu SH,Yang P,Sun T,Qi Q,Wang XQ,Xu DL,Chen XM.2016.Identification and evaluation of reference genes in the Chinese white wax scale insect Ericerus pela.Springer Plus,5:791
Yuan M,Lu Y,Zhu X,Wan H,Shakeel M,Zhan S,Jin BR,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(1):e86503
Zhang SD,An SH,Li Z,Wu FM,Yang QB,Liu YC,Cao JJ,Zhang HJ,Zhang QW,Liu XX.2015.Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae).Gene,555(2):393-402
Zhou X,Liao WJ,Liao JM,Liao P,Lu H.2015.Ribosomal proteins:functions beyond the ribosome.Journal of Molecular Cell Biology,7(2):92-104
Arya SK,Jain G,Upadhyay SK,Sarita,Singh H,Dixit S,Verma PC.2017.Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.Scientific Reports,7(1):13520
Bansal R,Mamidala P,Mian MA,Mittapalli O,Michel AP.2012.Validation of reference genes for gene expression studies in Aphis glycines(Hemiptera:Aphididae).Journal of Economic Entomology,105(4):1432-1438
Bustin SA,Benes V,Garson JA,Hellemans J,Huggett J,Kubista M,Mueller R,Nolan T,Pfaffl MW,Shipley GL,et al.2009.The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments.Clinical Chemistry,55(4):611-622
Chandrashekara KN,Kumar RR,Kumar V,Banerjee D,Kundu S,Ghosh B,Tyagi K.2015.DNA barcoding reveals host associated genetic diversity of‘tea mosquito bug’Helopeltis theivora(Miridae:Heteroptera)from India.Journal of Asia-Pacific Entomology,18(3):541-545
Chen F,Lu YY.2014.Selection of reference genes in Phenacoccus solenopsis(Hemiptera:Pseudococcidae)under heat stress.Acta Entomologica Sinica,57(10):1146-1154(in Chinese)[陈芳,陆永跃.2014.热胁迫下棉花粉蚧内参基因的筛选.昆虫学报,57(10):1146-1154]
Chen H,Yang ZQ,Hu Y,Tan JH,Jia J,Xu HL,Chen XH.2016.Reference genes selection for quantitative gene expression studies in Pinus massoniana L.Trees,30(3):685-696
Concha C,Edman RM,Belikoff EJ,Schiemann AH,Carey B,Scott MJ.2012.Organization and expression of the Australian sheep blowfly(Lucilia cuprina)hsp23,hsp24,hsp70 and hsp83 genes.Insect Molecular Biology,21(2):169-180
Dai TM,LüZC,Liu WX,Wan FH.2017.Selection and validation of reference genes for qRT-PCR analysis during biological invasions:the thermal adaptability of Bemisia tabaci MED.PLoSONE,12(3):e0173821
Dzaki N,Ramli KN,Azlan A,Ishak IH,Azzam G.2017.Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.Scientific Reports,7:43618
Ibanez F,Tamborindeguy C.2016.Selection of reference genes for expression analysis in the potato psyllid,Bactericera cockerelli.Insect Molecular Biology,25(3):227-238
Jian B,Liu B,Bi YR,Hou WS,Wu CX,Han TF.2008.Validation of internal control for gene expression study in soybean by quantitative real-time PCR.BMC Molecular Biology,9:59
Koramutla MK,Aminedi R,Bhattacharya R.2016.Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid,Lipaphis erysimi(Kalt).Scientific Reports,6:25883
Ladror DT,Frey BL,Scalf M,Levenstein ME,Artymiuk JM,Smith LM.2014.Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions.Biochemical and Biophysical Research Communications,445(3):535-541
Liang P,Guo Y,Zhou X,Gao X.2014.Expression profiling in Bemisia tabaci under insecticide treatment:indicating the necessity for custom reference gene selection.PLoS ONE,9(1):e87514
Liu GQ,Qiu XH,Cao L,Zhan ZB,Han RC.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(7):e0159060
Lu Y,Yuan M,Gao X,Kang T,Zhan S,Wan H,Li J.2013.Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura(Lepidoptera:Noctuidae).PLoS ONE,8(7):e68059
Luo YM,Jin QA.1991.A study on the mosquito bug infesting cashew in Hainan Island.Acta Entomologica Sinica,34(1):60-67(in Chinese)[罗永明,金启安.1991.海南岛腰果角盲蝽的研究.昆虫学报,34(1):60-67]
Nagy NA,Németh Z,Juhász E,Póliska S,Rácz R,Kosztolányi A,Barta Z.2017.Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle,Lethrus apterus(Coleoptera:Geotrupidae).PeerJ,5:e4047
Pan HP,Yang XW,Bidne K,Hellmich RL,Siegfried BD,Zhou XG,Zhang YJ.2015.Selection of reference genes for RT-qPCR analysis in the monarch butterfly,Danaus plexippus(L.),a migrating bio-indicator.PLoS ONE,10(6):e0129482
Pfaffl MW,Tichopad A,Prgomet C,Neuvians TP.2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations.Biotechnology Letters,26(6):509-515
P?achetka-Bo?ek A,Augustyniak M.2017.Evaluation of candidate reference genes for quantitative gene expression analysis in Spodoptera exigua after long-time exposure to cadmium.Scientific Reports,7(1):8338
Ponton F,Chapuis MP,Pernice M,Sword GA,Simpson SJ.2011.Evaluation of potential reference genes for reverse transcription-qP-CR studies of physiological responses in Drosophila melanogaster.Journal of Insect Physiology,57(6):840-850
Roy S,Muraleedharan N,Mukhapadhyay A,Handique G.2015.The tea mosquito bug,Helopeltis theivora Waterhouse(Heteroptera:Miridae):its status,biology,ecology and management in tea plantations.International Journal of Pest Management,61(3):179-197
Scharlaken B,de Graaf DC,Goossens K,Brunain M,Peelman LJ,Jacobs FJ.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,8:33
Shen GM,Jiang HB,Wang XN,Wang JJ.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
Shu BS,Zhang JJ,Cui GF,Sun RR,Sethuraman V,Yi X,Zhong GH.2018.Evaluation of reference genes for real-time quantitative PCR analysis in larvae of Spodoptera litura exposed to azadirachtin stress conditions.Frontiers in Physiology,9:372
Silver N,Best S,Jiang J,Thein SL.2006.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.BMC Molecular Biology,7:33
Simmons J,D’Souza O,Rheault M,Donly C.2013.Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.Insect Molecular Biology,22(1):62-71
Srikumar KK,Bhat PS.2013.Biology of the tea mosquito bug(Helopeltis theivora Waterhouse)on Chromolaena odorata(L.)R.M.King&H.Rob.Chilean Journal of Agricultural Research,73(3):309-314
Su L,Meng JY,Zhu JM,Zhang CY.2018.Cloning and expression of Hsp90 gene from green peach aphid Myzus persicae under UV-Bstress.Journal of Plant Protection,45(6):1267-1273(in Chinese)[苏丽,孟建玉,朱佳敏,张长禹,2018.烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析.植物保护学报,45(6):1267-1273]
Svitkin YV,Pause A,Haghighat A,Pyronnet S,Witherell G,Belsham GJ,Sonenberg N.2001.The requirement for eukaryotic initiation factor 4A(eIF4A)in translation is in direct proportion to the degree of mRNA 5’secondary structure.RNA,7(3):382-394
Taylor S,Wakem M,Dijkman G,Alsarraj M,Nguyen M.2010.A practical approach to RT-qPCR-publishing data that conform to the MIQE guidelines.Methods,50(S):1-5
Teng XL,Zhang Z,He GL,Yang LW,Li F.2012.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects.Journal of Insect Science,12:60
Vandesompele J,de Preter K,Pattyn F,Poppe B,van Roy N,de Paepe A,Speleman F.2002.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biology,3(7):research0034
Volkov RA,Panchuk II,Sch?ffl F.2003.Heat-stress-dependency and developmental modulation of gene expression:the potential of house-keeping genes as internal standards in m RNA expression profiling using real-time RT-PCR.Journal of Experimental Botany,54(391):2343-2349
Wang B,Du HH,Yao ZP,Ren C,Ma L,Wang J,Zhang H,Ma H.2018.Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses.Physiology and Molecular Biology of Plant,24(3):455-463
Wang Z,Meng QQ,Liu AQ,Sang LW,Sun SW,Gou YF,Gao SF.2017.Observation of the sensilla on antenna and propodium of Helopeltis theivora Waterhouse(Hemiptera:Mirada)with scanning electron microscope.Chinese Journal of Tropical Crops,38(11):2165-2170(in Chinese)[王政,孟倩倩,刘爱勤,桑利伟,孙世伟,苟亚峰,高圣风.2017.茶角盲蝽触角和前足感器的扫描电镜观察.热带作物学报,38(11):2165-2170]
Wu S,Zhang XF,He YQ,Shuai JB,Chen XM,Ling EJ.2010.Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development.Developmental&Comparative Immunology,34(11):1191-1198
Xie FL,Sun GL,Stiller JW,Zhang BH.2011.Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database.PLoS ONE,6(11):e26980
Yang L,Hu XJ,Xu ZF,He L,Xiao W.2017.Screening of reference genes for qRT-PCR in Conogethes punctiferalis(Lepidoptera:Crambidae).Acta Entomologica Sinica,60(11):1266-1277(in Chinese)[杨苓,胡晓静,徐志峰,何林,肖伟.2017.桃蛀螟实时荧光定量PCR内参基因的筛选.昆虫学报,60(11):1266-1277]
Yu SH,Yang P,Sun T,Qi Q,Wang XQ,Xu DL,Chen XM.2016.Identification and evaluation of reference genes in the Chinese white wax scale insect Ericerus pela.Springer Plus,5:791
Yuan M,Lu Y,Zhu X,Wan H,Shakeel M,Zhan S,Jin BR,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(1):e86503
Zhang SD,An SH,Li Z,Wu FM,Yang QB,Liu YC,Cao JJ,Zhang HJ,Zhang QW,Liu XX.2015.Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera(Lepidoptera:Noctuidae).Gene,555(2):393-402
Zhou X,Liao WJ,Liao JM,Liao P,Lu H.2015.Ribosomal proteins:functions beyond the ribosome.Journal of Molecular Cell Biology,7(2):92-104