2022 03 v.33;No.107 443-452
褐飞虱若虫、成虫期荧光定量PCR内参基因的筛选
基金项目(Foundation):
国家自然科学基金项目(No.31672026,U21A20223,32011530117);;
浙江省科技计划项目(No.LY20C140005,2019C02015,2022C02047);;
中国计量大学科研标志性发展专项项目(No.2020YW27)
邮箱(Email):
haopy@cjlu.edu.cn;
DOI:
中文作者单位:
中国计量大学生命科学学院浙江省生物计量及检验检疫技术重点实验室;
摘要(Abstract):
目的:筛选适用于褐飞虱Nilaparvata lugens成虫期目的基因表达水平检测的内参基因。方法:根据褐飞虱转录组数据初步筛选候选内参基因，采用RT-qPCR技术检测候选内参基因RPS11、β-actin和18S(18S rRNA)在褐飞虱不同发育阶段的表达水平，分别按照若虫阶段、成虫阶段、若虫-成虫阶段(包含若虫期和成虫期)3种情况分析内参基因在不同发育阶段的表达稳定性，用geNorm、NormFinder、BestKeeper和ΔCt 4种方法对数据进行处理，并用RefFinder对以上4种方法进行内参基因稳定性综合分析。结果:在褐飞虱若虫阶段，geNorm、NormFinder、BestKeeper和ΔCt 4种方法均表明RPS11基因的稳定性最好；在褐飞虱成虫阶段和若虫-成虫阶段，ΔCt和NormFinder方法分析表明，18S作为内参基因更稳定，而BestKeeper分析显示RPS11基因比18S稳定，geNorm分析表明RPS11与18S基因稳定性相同，4种方法均表明β-actin基因的稳定性最差。RefFinder综合以上4种分析方法，结果显示3个内参基因稳定性由强到弱依次为：18S、RPS11和β-actin。分别以RPS11、β-actin和18S为内参基因，检测NlATG3基因在褐飞虱成虫阶段羽化后不同天数的相对表达量，发现NlATG3基因在褐飞虱成虫羽化后不同天数的表达规律因内参基因的不同而出现差异。结论:内参基因RPS11在褐飞虱若虫时期表达稳定，但18S更适用于检测褐飞虱成虫阶段羽化后不同天数目的基因的表达水平。
关键词(KeyWords):
褐飞虱;;发育阶段;;内参基因;;稳定性
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[2] ZHANG L,ZHANG Q L,WANG X T,et al.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[J].Ecology and Evolution,2017,7(21):9054-9065.
[3] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[4] KANG Z W,LIU F H,TIAN H G,et al.Evaluation of the reference genes for expression analysis using quantitative real-time polymerase chain reaction in the green peach aphid,Myzus persicae[J].Insect Science,2017,24(2):222-234.
[5] SAGRI E,KOSKINIOTI P,GREGORIOU M E,et al.Housekeeping in tephritid insects:the best gene choice for expression analyses in the medfly and the olive fly[J].Scientific Reports,2017,7:45634.
[6] NICHOLLS C,LI H,LIU J P.GAPDH:A common enzyme with uncommon functions[J].Clinical and Experimental Pharmacology and Physiology,2012,39(8):674-679.
[7] ZHOU X,LIAO W J,LIAO J M,et al.Ribosomal proteins:functions beyond the ribosome[J].Journal of Molecular Cell Biology,2015,7(2):92-104.
[8] ZHOU L,MENG J Y,RUAN H Y,et al.Expression stability of candidate RT-PCR housekeeping genes in Spodoptera frugiperda (Lepidoptera:Noctuidae)[J].Archives of Insect Biochemistry and Physiology,2021,108(1):e21831.
[9] CHOUDHURY A,VERMA S,MUTHAMILARASAN M,et al.Identification of suitable reference genes for expression profiling studies using qRT-PCR in an important insect pest,Maruca vitrata[J].Molecular Biology Reports,2021,48(11):7477-7485.
[10] HU Z Y,DENG Y C,ZHANG X L,et al.Selection and validation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) in silkworm infected with Bombyx mori bidensovirus[J].Biologia,2018,73(9):897-906.
[11] WANG G H,XIA Q Y,CHENG D J,et al.Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR[J].Insect Science,2008,15(5):405-413.
[12] KIM Y,KIM Y,KIM Y H.Evaluation of reference genes for gene expression studies using quantitative real-time PCR in Drosophila melanogaster after chemical exposures[J].Journal of Asia-Pacific Entomology,2020,23(2):385-394.
[13] LI R M,XIE W,WANG S L,et al.Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly,Bemisia tabaci (Hemiptera:Aleyrodidae)[J].PLoS One,2013,8(1):e53006.
[14] BOTTRELL D G,SCHOENLY K G.Resurrecting the ghost of green revolutions past:The brown planthopper as a recurring threat to high-yielding rice production in tropical Asia[J].Journal of Asia-Pacific Entomology,2012,15(1):122-140.
[15] JIANG L B,CHENG J,ZHU Z F,et al.Impact of day intervals on sequential infestations of the rice leaffolder Cnaphalocrocis medinalis (Guenée) (Lepidoptera:Pyralidae) and the white-backed rice planthopper Sogatella furcifera (Horváth) on rice grain damage[J].International Journal of Insect Science,2014,6(6):23-31.
[16] BAE S H,PATHAK M.Life history of Nilaparvata lugens (Homoptera:Delphacidae) and susceptibility of rice varieties to its attacks[J].Annals of the Entomological Society of America,1970,63(1):149-155.
[17] BACKUS E A,SERRANO M S,RANGER C M.Mechanisms of hopperburn:An overview of insect taxonomy,behavior,and physiology[J].Annual Review of Entomology,2005,50(1):125-151.
[18] 程家安，祝增荣.中国水稻病虫草害治理60年：问题与对策[J].植物保护学报，2017,44(6):885-895.CHENG J A,ZHU Z R.Development of rice pest management in the past 60 years in china:problems and strategies[J].Acta Phytophylacica Sinica,2017,44(6):885-895.
[19] WAN P J,YANG L,YUAN S Y,et al.RNA interference-aided knockdown of a putative saccharopine dehydrogenase leads to abnormal ecdysis in the brown planthopper,Nilaparvata lugens (St?l) (Hemiptera:Delphacidae)[J].Bulletin of Entomological Research,2015,105(4):390-398.
[20] JIANG J J,HUANG L F,CHEN H S,et al.Identification of reference genes and expression analysis of heat shock protein genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae),after exposure to heat stress[J].Acta Entomologica Sinica,2015,32(5):633-639.
[21] YUAN M,LU Y H,ZHU X,et al.Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper,Nilaparvata lugens (Hemiptera:Delphacidae) using reverse-transcription quantitative PCR[J].PLoS One,2014,9(1):e86503.
[22] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J].Methods,2001,25(4):402-408.
[23] YE C L,FENG Y L,YU F F,et al.RNAi-mediated silencing of the autophagy-related gene NlATG3 inhibits survival and fecundity of the brown planthopper,Nilaparvata lugens[J].Pest Management Science,2021,77(10):4658-4668.
基本信息:
DOI：
中图分类号:S435.112.3
引用信息:
[1]矫启启,叶成龙,冯娅琳等.褐飞虱若虫、成虫期荧光定量PCR内参基因的筛选[J].中国计量大学学报,2022,33(03):443-452.
基金信息:
国家自然科学基金项目(No.31672026,U21A20223,32011530117);; 浙江省科技计划项目(No.LY20C140005,2019C02015,2022C02047);; 中国计量大学科研标志性发展专项项目(No.2020YW27)
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