银杏实时荧光定量PCR分析中内参基因的选择与验证
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摘要
为了筛选出银杏中稳定的内参基因用于实时荧光定量PCR (qRT-PCR)分析,本研究以银杏(Ginkgo biloba)的不同组织,不同发育时期雌株、雄株叶片为材料,使用3种软件geNorm, NormFinderand和Bestkeeper综合评估银杏中常见的7个内参基因(GbACT、GbUBQ、GbEF-1α1、GbEF-1α2、GbGAPDH、Gb18S和Gb26S)的稳定性,并用查尔酮合酶基因(Chalcone synthase, CHS)对内参基因稳定性进行验证。结果表明,候选基因的稳定性各异, GbUBQ是所有测试样品和不同发育时期雄株叶片中表达最稳定的基因, GbACT是不同时期雌株叶片表达最稳定的内参基因, GbGAPDH是不同组织中表达最稳定的基因。
In order to screen out stable reference genes in Ginkgo biloba for real-time quantitative PCR(qRTPCR) analysis, we used different tissues, the leaves of female plants and male plants at different developmental stages of G. biloba as materials. The stability of the seven genes(GbACT, GbUBQ, GbEF-1α1, GbEF-1α2, GbGAPDH, Gb18 S, Gb26 S) were evaluated by three software(geNorm, NormFinder and Bestkeeper) and GbCHS was used to verify it. The results showed that candidate genes varied in stability. GbUBQ was the most stable reference gene in all samples and male leaves of different periods. GbACT was the most stable reference gene in female leaves in different periods. GbGAPDH was the most stable gene in different tissues.
In order to screen out stable reference genes in Ginkgo biloba for real-time quantitative PCR(qRTPCR) analysis, we used different tissues, the leaves of female plants and male plants at different developmental stages of G. biloba as materials. The stability of the seven genes(GbACT, GbUBQ, GbEF-1α1, GbEF-1α2, GbGAPDH, Gb18 S, Gb26 S) were evaluated by three software(geNorm, NormFinder and Bestkeeper) and GbCHS was used to verify it. The results showed that candidate genes varied in stability. GbUBQ was the most stable reference gene in all samples and male leaves of different periods. GbACT was the most stable reference gene in female leaves in different periods. GbGAPDH was the most stable gene in different tissues.
引文
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Jiang TT,Gao YH,Tong ZK(2015).Selection of reference genes for quantitative real-time PCR in Lycoris.Acta Hortic Sin,42(6):1129-1138(in Chinese with English abstract)[蒋婷婷,高燕会,童再康(2015).石蒜属植物实时荧光定量PCR内参基因的选择.园艺学报,42(6):1129-1138]
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Lin YL(2011).Study on cloning,expression and regulation of SOD gene family during somatic embryogenesis in Dimocarpus longan Lour.(dissertation).Fuzhou:Fujian Agriculture and Forestry University(in Chinese with English abstract)[林玉玲(2011).龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究(学位论文).福州:福建农林大学]
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Vandesompele J,De Preter K,Pattyn F,et al(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):research0034
Wang H,Cao F,Zhang W,et al(2013).Cloning and expression of Stearoyl-ACP desaturase and two oleate desaturases genes from Ginkgo biloba L.Plant Mol Biol Report,31(3):633-648
Wang YJ,Dong L,Zhang C,et al(2012).Reference gene selection for real-time quantitative PCR normalization in tree peony(Paeonia suffruticosa Andr.).J Agric Biotech,20(5):521-528(in Chinese with English abstract)[王彦杰,董丽,张超等(2012).牡丹实时定量PCR分析中内参基因的选择.农业生物技术学报,20(5):521-528]
Wen L,Tan B,Guo WW(2012).Estimating transgene copy number in precocious trifoliate orange by TaqMan real-time PCR.Plant Cell Tiss Org Cult,109(2):363-371
Xu F(2005).Molecular cloning and expression of chalcone synthase gene and phenylalanine ammonia-lyase gene from Ginkgo biloba(dissertation).Wuhan:Huazhong Agriculture University(in Chinese with English abstract)[许锋(2005).银杏查尔酮合成酶基因和苯丙氨酸解氨酶基因的克隆及表达(学位论文).武汉:华中农业大学]
Yoo WG,Kim TI,Li S,et al(2009).Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR.Parasitol Res,104(2):321-328
Zhang JY,Huang SN,Wang T,et al(2018).Screening of reference of genes for reverse transcription quantitative real-time PCR in Atinidia diliciosa.Acta Agric Shanghai,34(1):84-88(in Chinese with English abstract)[张计育,黄胜男,王涛等(2018).金魁猕猴桃RT-qPCR内参基因的筛选.上海农业学报,34(1):84-88]
Andersen CL,Jensen JL,?rntoft TF et al(2004).Normalization of real-time quantitative reverse transcription-PCRdata:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets.Cancer Res,64(15):5245-5250
Artico S,Nardeli SM,Brilhante O,et al(2010).Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.BMC Plant Biol,10:49
Brunner AM,Yakovlev IA,Strauss SH(2004).Validating internal controls for quantitative plant gene expression studies.BMC Plant Biol,4(1):14
Cao FL(2007).Chinese Ginkgo.Beijing:China Forestry Publishing House,14-27(in Chinese with English abstract)[曹福亮(2007).中国银杏志.北京:中国林业出版社,14-27]
Chang E,Shi S,Liu J,et al(2012).Selection of reference genes for quantitative gene expression studies in Platycladus orientalis(Cupressaceae)using real-time PCR.PLoS One,7(3):e33278
Cheng H,Li LL,Cheng SY,et al(2013).Molecular cloning and characterization of three genes encoding dihydroflavonol-4-reductase from Ginkgo biloba in anthocyanin biosynthetic pathway.PLoS One,8(8):e72017
Czechowski T,Stitt M,Altmann T,et al(2005).Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis.Plant Physiol,139(1):5-17
Gutierrez L,Mauriat M,Guénin S,et al(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 Biotech J,6(6):609-618
Jain M,Nijhawan A,Tyagi AK,et al(2006).Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR.Biochem Biophys Res Commun,345(2):646-651
Jiang T,Su Q,An LJ(2015).Screening and validation of reference genes of qPCR in maize under multiple stresses.Plant Physio J,51(9):1457-1464(in Chinese with English abstract)[姜婷,苏乔,安利佳(2015).多重胁迫下玉米实时定量PCR内参基因的筛选与验证.植物生理学报,51(9):1457-1464]
Jiang TT,Gao YH,Tong ZK(2015).Selection of reference genes for quantitative real-time PCR in Lycoris.Acta Hortic Sin,42(6):1129-1138(in Chinese with English abstract)[蒋婷婷,高燕会,童再康(2015).石蒜属植物实时荧光定量PCR内参基因的选择.园艺学报,42(6):1129-1138]
Kong Q,Yuan J,Gao L,et al(2014).Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.PLoS One,9(2):e90612
Kulcheski FR,Marcelino-Guimaraes FC,Nepomuceno AL,et al(2010).The ues of microRNAs as reference genes for quantitative polymerase chain reaction in soybean.Anal Biochem,406(2):185-192
Libault M,Thibivilliers S,Bilgin DD,et al(2008).Identification of four soybean reference genes for gene expression normalization.Plant Geno,1(1):44-54
Lin YL(2011).Study on cloning,expression and regulation of SOD gene family during somatic embryogenesis in Dimocarpus longan Lour.(dissertation).Fuzhou:Fujian Agriculture and Forestry University(in Chinese with English abstract)[林玉玲(2011).龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究(学位论文).福州:福建农林大学]
Mafra V,Kubo KS,Alves-Ferreira M,et al(2012).Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.PLoS One,7(2):e31263
Nicot N,Hausman JF,Hoffmann L,et al(2005).Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.Plant Biotechnol J,56(421):12387
Pfaffl MW,Tichopad A,Prgomet C,Neuvians TP(2004).Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeperExcel-based tool using pair-wise correlations.Biotechnol Lett,26(6):509-515
Remans T,Smeets K,Opdenakker K,et al(2008).Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations.Planta,227(6):1343-1349
Thomas C,Meyer D,Wolff M,et al(2003).Molecular characterization and spatial expression of the sunflower ABP1gene.Plant Molecular Biol,52(5):1025-1036
Vandesompele J,De Preter K,Pattyn F,et al(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol,3(7):research0034
Wang H,Cao F,Zhang W,et al(2013).Cloning and expression of Stearoyl-ACP desaturase and two oleate desaturases genes from Ginkgo biloba L.Plant Mol Biol Report,31(3):633-648
Wang YJ,Dong L,Zhang C,et al(2012).Reference gene selection for real-time quantitative PCR normalization in tree peony(Paeonia suffruticosa Andr.).J Agric Biotech,20(5):521-528(in Chinese with English abstract)[王彦杰,董丽,张超等(2012).牡丹实时定量PCR分析中内参基因的选择.农业生物技术学报,20(5):521-528]
Wen L,Tan B,Guo WW(2012).Estimating transgene copy number in precocious trifoliate orange by TaqMan real-time PCR.Plant Cell Tiss Org Cult,109(2):363-371
Xu F(2005).Molecular cloning and expression of chalcone synthase gene and phenylalanine ammonia-lyase gene from Ginkgo biloba(dissertation).Wuhan:Huazhong Agriculture University(in Chinese with English abstract)[许锋(2005).银杏查尔酮合成酶基因和苯丙氨酸解氨酶基因的克隆及表达(学位论文).武汉:华中农业大学]
Yoo WG,Kim TI,Li S,et al(2009).Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR.Parasitol Res,104(2):321-328
Zhang JY,Huang SN,Wang T,et al(2018).Screening of reference of genes for reverse transcription quantitative real-time PCR in Atinidia diliciosa.Acta Agric Shanghai,34(1):84-88(in Chinese with English abstract)[张计育,黄胜男,王涛等(2018).金魁猕猴桃RT-qPCR内参基因的筛选.上海农业学报,34(1):84-88]