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甜瓜实时荧光定量PCR分析中内参基因的筛选

章丽珍 韩晓云 吴菁华 GefuWANG-PRUSKI 张志忠

章丽珍,韩晓云,吴菁华,等. 甜瓜实时荧光定量PCR分析中内参基因的筛选 [J]. 福建农业学报,2020,35(11):1179−1187 doi: 10.19303/j.issn.1008-0384.2020.11.002
引用本文: 章丽珍,韩晓云,吴菁华,等. 甜瓜实时荧光定量PCR分析中内参基因的筛选 [J]. 福建农业学报,2020,35(11):1179−1187 doi: 10.19303/j.issn.1008-0384.2020.11.002
ZHANG L Z, HAN X Y, WU J H, et al. Reference Gene Selection for RT-qPCR Analysis on Cucumis melo [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1179−1187 doi: 10.19303/j.issn.1008-0384.2020.11.002
Citation: ZHANG L Z, HAN X Y, WU J H, et al. Reference Gene Selection for RT-qPCR Analysis on Cucumis melo [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1179−1187 doi: 10.19303/j.issn.1008-0384.2020.11.002

甜瓜实时荧光定量PCR分析中内参基因的筛选

doi: 10.19303/j.issn.1008-0384.2020.11.002
基金项目: 福建省闽江学者科研基金(116-114120019);福建农林大学创新专项基金(CXZX2016108、CXZX2017168)
详细信息
    作者简介:

    章丽珍(1994−),女,硕士研究生,研究方向:农艺与种业(E-mail:651260775@qq.com

    通讯作者:

    张志忠(1976−),男,博士,副教授,研究方向:园艺植物逆境生理和生物技术(E-mail:zeada2001@163.com

  • 中图分类号: S 652

Reference Gene Selection for RT-qPCR Analysis on Cucumis melo

  • 摘要:   目的  筛选可分别在甜瓜不同组织器官和不同胁迫处理下稳定表达的内参基因,用于对靶基因表达量的实时荧光定量分析,保证相关试验的准确性及可靠性。  方法  以甜瓜品种新银辉为试验材料,通过实时荧光定量PCR技术分析18s rRNATUAEF1aActin1Actin2Actin3Actin4CYCUBI-ep共9个候选内参基因在甜瓜不同组织器官及不同胁迫处理下的表达稳定性,包括甜瓜根、叶、种子和果实4种不同组织材料,以及水分胁迫、肉桂酸胁迫、盐碱胁迫和ABA胁迫4种处理。同时使用Best-Keeper、Norm Finder和ge Norm软件对9个候选内参基因进行稳定性分析。  结果  对不同组织器官而言,Best-Keeper评估排名前5的内参基因依次为CYC18s rRNAUBI-epEF1aTUA;Norm Finder计算排名前5的内参基因依次为EF1aUBI-epActin4CYCActin3;ge Norm分析排名前5的基因依次为Actin4=Actin3Actin1EF1aUBI-ep。不同胁迫处理中,Best-Keeper计算排名前5的基因依次为18s rRNAActin3Actin4EF1aUBI-ep;Norm Finder分析排名前5的基因依次为EF1aUBI-epActin4CYC18s rRNA;ge Norm分析排名前5的基因依次为EF1a=UBI-epActin4CYCActin1。总体而言EF1a在不同组织器官和不同胁迫处理中的综合排名均较为稳定;Actin4Actin3Actin1EF1a是不同组织器官中较为稳定的内参基因组合;EF1aUBI-ep是4种胁迫条件下较稳定的内参基因组合。  结论  EF1a在甜瓜不同组织器官及不同胁迫条件下均可稳定表达,是较为合适的内参基因;同时可通过设置双内参基因进一步降低试验误差。
  • 图  1  9个候选内参基因的引物扩增特异性与扩增长度

    注:M:Marker;1~9分别为内参基因Actin1Actin2、Actin3Actin4、CYCUBI-epTUA18s rRNAEF1a

    Figure  1.  Amplification specificities and lengths of 9 candidate reference genes

    Note: M: marker; 1–9: candidate reference genes, Actin1, Actin2, Actin3, Actin4, CYC,UBI-ep, TUA, 18s rRNA, and EF1a, respectively.

    图  2  9个候选内参基因的熔解曲线

    Figure  2.  Melting curves for 9 candidate reference genes

    图  3  9个候选内参基因Ct值箱线图

    注:箱线图箱体代表Ct值主要集中范围;上边表示上四分线;中线表示中位数;下边表示下四分线;上边缘表示本组数据最大值;下边缘表示本组数据最小值。

    Figure  3.  Box plot of Ct values of 9 reference genes

    Note: Box: range of concentrated Ct values; top line: upper quarter line; middle line: median value; bottom line: lower quarter line; top edge: maximum value of group; bottom edge: minimum of group.

    图  4  9个候选内参基因的配对数变异指数

    Figure  4.  Calculated pairwise variations (V) of 9 candidate reference genes

    表  1  9个候选内参基因RT-qPCR引物序列及其扩增效率

    Table  1.   RT-qPCR primer sequences and amplification efficiency of 9 candidate reference genes

    登录号
    GenBank ID
    基因
    Gene
    引物序列(5′-3′)
    Primer sequence F/R(5′-3′)
    产物大小
    Product size/bp
    扩增效率
    Efficiency(E)
    AF206894.118s rRNATCTGCCCGTTGCTCTGATG1302.05
    TCACCCGTCACCACCATAG
    XM_008456199.2TUAACGCTGTTGGTGGTGGTAC1062.06
    GAGAGGGGTAAACAGTGAATC
    XM_008459007.2EF1aACTGTGCTGTCCTCATTATTG981.98
    AGGGTGAAAGCAAGAAGAGC
    LN713255.1CYCGATGGAGCTCTACGCCGATGTC1531.97
    CCTCCCTGGCACATGAAATTAG
    NM_001282241.1UBI-epCACCAAGCCCAAGAAGATC2202.10
    TAAACCTA ATCACCACCAGC
    XM_008442791.2Actin1TTCTGGTGATGGTGTGAGTC1492.09
    GGCAGTGGTGGTGAACATG
    XM_008449644.2Actin2GAAGGAATAACCACGCTCAG1172.02
    ACACAGTTCCCATCTACGAG
    XM_008449644.2Actin3GGCAGTGGTGGTGAACATG1491.99
    TTCTGGTGATGGTGTGAGTC
    LN713262.1Actin4TGGAAGCTGCAGGAATCCACGA1652.04
    TGCTGGGAGCAAGGGCTGTG
    下载: 导出CSV

    表  2  Best-Keeper软件分析9个候选内参基因稳定性

    Table  2.   Stabilities of 9 reference genes analyzed by BestKeeper software

    样品分组  
    Sample group  
    基因
    Gene
    几何平均值
    Geometric
    mean value
    最大值
    Max
    最小值
    Mix
    标准差
    Stardand
    deviaton(SD)
    变异差
    Coefficient
    ofvariation(CV)
    稳定性排名
    Ranking of
    stable value
    不同组织器官
    Different tissues
    CYC 20.88 21.55 19.72 0.57 2.72 1
    18s rRNA 10.21 11.46 8.90 0.77 7.55 2
    UBI-ep 20.12 22.10 18.69 0.94 4.67 3
    EF1a 20.42 22.03 19.11 0.95 4.65 4
    TUA 22.69 24.80 21.10 0.98 4.31 5
    Actin1 21.92 23.98 19.23 1.37 6.21 6
    Actin4 20.63 23.14 18.99 1.38 6.65 7
    Actin3 21.61 24.03 19.39 1.50 6.92 8
    Actin2 31.38 33.95 29.40 1.86 5.92 9
    不同胁迫处理
    Different stress conditions
    18s rRNA 11.82 12.31 11.16 0.22 1.86 1
    Actin3 22.85 23.30 21.54 0.37 1.62 2
    Actin4 21.85 22.77 21.23 0.43 1.97 3
    EF1a 21.24 22.20 20.36 0.46 2.18 4
    UBI-ep 21.38 22.29 20.49 0.46 2.14 5
    Actin1 23.20 24.22 22.28 0.54 2.35 6
    CYC 18.87 19.89 17.95 0.59 3.44 7
    TUA 22.05 23.35 21.27 0.60 2.71 8
    Actin2 36.68 35.63 28.84 1.86 5.67 9
    下载: 导出CSV

    表  3  Norm Finder和ge Norm软件分析候9个选内参基因稳定性

    Table  3.   Stabilities of 9 reference genes analyzed by NormFinder and geNorm software

    样品分组  
    Sample group  
    Norm Finder ge Norm
    基因
    Gene
    稳定值
    Stability value
    稳定性排名
    Ranking of stable value
    基因
    Gene
    稳定值
    Stability value
    稳定性排名
    Ranking of stable value
    不同组织器官
    Different tissues
    EF1a 0.107 1 Actin4 0.526 1
    UBI-ep 0.153 2 Actin3 0.526 2
    Actin4 0.228 3 Actin1 0.640 3
    CYC 0.243 4 EF1a 0.794 4
    Actin3 0.291 5 UBI-ep 0.894 5
    18s rRNA 0.315 6 18s rRNA 0.961 6
    Actin1 0.319 7 CYC 1.101 7
    TUA 0.345 8 TUA 1.318 8
    Actin2 0.423 9 Actin2 1.462 9
    不同胁迫处理
    Different stress conditions
    EF1a 0.023 1 EF1a 0.138 1
    UBI-ep 0.053 2 UBI-ep 0.138 2
    Actin4 0.059 3 Actin4 0.145 3
    CYC 0.078 4 CYC 0.207 4
    18s rRNA 0.128 5 TUA 0.242 5
    TUA 0.128 6 18s rRNA 0.321 6
    Actin1 0.214 7 Actin1 0.373 7
    Actin3 0.236 8 Actin3 0.445 8
    Actin2 0.264 9 Actin2 0.781 9
    下载: 导出CSV

    表  4  9个选内参基因稳定性综合排名

    Table  4.   Overall ranking on expression stabilities of 9 reference genes

    样品分组
    Sample group
    基因
    Gene
    Best-KeeperNorm-Finderge Norm几何平均值
    Geometric mean value
    综合排名
    Comprehensive rankings
    不同组织器官
    Different tissues
    EF1a4142.5201
    Actin47312.7592
    CYC1473.0373
    UBI-ep3253.1074
    18s rRNA2664.1605
    Actin38524.3096
    Actin16735.0137
    TUA5886.8408
    Actin29999.0009
    不同胁迫处理
    Different stress conditions
    EF1a4111.5871
    UBI-ep5222.7142
    Actin43333.0003
    18s rRNA1563.1074
    CYC7444.8205
    Actin32885.0406
    TUA8656.2147
    Actin16776.6498
    Actin29999.0009
    下载: 导出CSV
  • [1] GINZINGER D G. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream [J]. Experimental Hematology, 2002, 30(6): 503−512. doi: 10.1016/S0301-472X(02)00806-8
    [2] ZHANG Z Z, FAN J R, WU J H, et al. Alleviating effect of silicon on melon seed germination under autotoxicity stress [J]. Ecotoxicology and Environmental Safety, 2020, 188: 109901. doi: 10.1016/j.ecoenv.2019.109901
    [3] PFAFFL M W, TICHOPAD A, PRGOMET C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations [J]. Biotechnology Letters, 2004, 26(6): 509−515. doi: 10.1023/B:BILE.0000019559.84305.47
    [4] VANDESOMPELE J, DE PRETER K, PATTYN F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J]. Genome Biology, 2002, 3(7): 1−12.
    [5] TANG X, ZHANG N, SI H J, et al. Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress [J]. Plant Methods, 2017, 13: 85. doi: 10.1186/s13007-017-0238-7
    [6] LUO H L, LUO L P, GUAN B C, et al. Evaluation of candidate reference genes for RT-qPCR in lily (Lilium brownii) [J]. The Journal of Horticultural Science and Biotechnology, 2014, 89(3): 345−351. doi: 10.1080/14620316.2014.11513089
    [7] 吕运舟, 董筱昀, 黄利斌. 黄山栾树实时荧光定量PCR内参基因的筛选 [J]. 分子植物育种, 2019, 17(2):553−560.

    LÜ Y Z, DONG X Y, HUANG L B. The screening of reference genes for real-time fluorescent quantitative PCR of Koelreuteria bipinnata [J]. Molecular Plant Breeding, 2019, 17(2): 553−560.(in Chinese)
    [8] 任锐, 戴鹏辉, 李萌, 等. 珙桐实时定量PCR内参基因的筛选及稳定性评价 [J]. 植物生理学报, 2016, 52(10):1565−1575.

    REN R, DAI P H, LI M, et al. Selection and stability evaluation of reference genes for real-time quantitative PCR in dove tree (Davidia involucrata) [J]. Plant Physiology Communications, 2016, 52(10): 1565−1575.(in Chinese)
    [9] GOPALAM R, RUPWATE S D, TUMANEY A W. Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica [J]. PLoS One, 2017, 12(11): e0186978. doi: 10.1371/journal.pone.0186978
    [10] 蒋婷婷, 高燕会, 童再康. 石蒜属植物实时荧光定量PCR内参基因的选择 [J]. 园艺学报, 2015, 42(6):1129−1138.

    JIANG T T, GAO Y H, TONG Z K. Selection of reference genes for quantitative real-time PCR in Lycoris [J]. Acta Horticulturae Sinica, 2015, 42(6): 1129−1138.(in Chinese)
    [11] 王彦杰, 董丽, 张超, 等. 牡丹实时定量PCR分析中内参基因的选择 [J]. 农业生物技术学报, 2012, 20(5):521−528. doi: 10.3969/j.issn.1674-7968.2012.05.008

    WANG Y J, DONG L, ZHANG C, et al. Reference gene selection for real-time quantitative PCR normalization in tree peony (Paeonia suffruticosa andr.) [J]. Journal of Agricultural Biotechnology, 2012, 20(5): 521−528.(in Chinese) doi: 10.3969/j.issn.1674-7968.2012.05.008
    [12] FAUSTO A K S, SILVA T D F, ROMANEL E, et al. microRNAs as reference genes for quantitative PCR in cotton [J]. PLoS One, 2017, 12(4): e0174722. doi: 10.1371/journal.pone.0174722
    [13] 潘红, 赖呈纯, 张静, 等. 不同光质条件下刺葡萄红色愈伤组织的RT-qPCR内参基因筛选 [J]. 应用与环境生物学报, 2019, 25(6):1407−1413.

    PAN H, LAI C C, ZHANG J, et al. Selection of reference genes for RT-qPCR from the red callus of Vitis davidii (Rom. Caill.) Fo(ë)x under different light qualities [J]. Chinese Journal of Applied & Environmental Biology, 2019, 25(6): 1407−1413.(in Chinese)
    [14] GONZÁLEZ-VERDEJO C I, DIE J V, NADAL S, et al. Selection of housekeeping genes for normalization by real-time RT–PCR: Analysis of Or-MYB1 gene expression in Orobanche ramosa development [J]. Analytical Biochemistry, 2008, 379(2): 176−181. doi: 10.1016/j.ab.2008.05.003
    [15] 史兴青. 甜瓜生长发育和胁迫条件下实时荧光定量PCR内参基因的筛选 [D]. 郑州: 河南农业大学, 2016.

    SHI X Q. Selection of suitable reference genes for quantitative real-time RT-PCR studies in Cucumis melo under growth and development process, biotic and abiotic stresses[D]. Zhengzhou: Henan Agricultural University, 2016. (in Chinese).
    [16] ANDERSEN C L, JENSEN J L, ØRNTOFT T F. 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 [J]. Cancer Research, 2004, 64(15): 5245−5250. doi: 10.1158/0008-5472.CAN-04-0496
    [17] SUDHAKAR REDDY P, SRINIVAS REDDY D, SIVASAKTHI K, et al. Evaluation of Sorghum [Sorghum bicolor (L.)] reference genes in various tissues and under abiotic stress conditions for quantitative real-time PCR data normalization [J]. Frontiers in Plant Science, 2016, 7: 529. doi: 10.3389/fpls.2016.00529
    [18] LI L, WANG K Y, ZHAO M Z, et al. Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots [J]. PLoS One, 2019, 14(12): e0226168. doi: 10.1371/journal.pone.0226168
    [19] 乔永刚, 王勇飞, 曹亚萍, 等. 药用蒲公英低温和高温胁迫下内参基因筛选与相关基因表达分析 [J]. 园艺学报, 2020, 47(6):1153−1164.

    QIAO Y G, WANG Y F, CAO Y P, et al. Reference genes selection and related genes expression analysis under low and high temperature stress in Taraxacum officinale [J]. Acta Horticulturae Sinica, 2020, 47(6): 1153−1164.(in Chinese)
    [20] 宋晓波, 常英英, 刘昊, 等. 核桃不定根发生阶段内参基因筛选与关键基因表达分析 [J]. 园艺学报, 2019, 46(10):1907−1918.

    SONG X B, CHANG Y Y, LIU H, et al. Reference gene selection and genes expression analysis during adventitious root formation in walnut [J]. Acta Horticulturae Sinica, 2019, 46(10): 1907−1918.(in Chinese)
    [21] 刘涛, 熊青, 许颖妍, 等. 夜香树花期荧光定量PCR内参基因的筛选 [J]. 植物科学学报, 2017, 35(4):534−542. doi: 10.11913/PSJ.2095-0837.2017.40534

    LIU T, XIONG Q, XU Y Y, et al. Selection of reference genes for qRT-PCR normalization in Cestrum nocturnum during flowering [J]. Plant Science Journal, 2017, 35(4): 534−542.(in Chinese) doi: 10.11913/PSJ.2095-0837.2017.40534
    [22] 胡宁宁, 郭慧琴, 李西良, 等. 羊草不同组织实时定量PCR 内参基因的筛选 [J]. 草业科学, 2017, 34(7):1434−1441. doi: 10.11829/j.issn.1001-0629.2016-0510

    HU N N, GUO H Q, LI X L, et al. Selection of reference genes for quantitative real-time PCR of Leymus chinensis in different tissues [J]. Pratacultural Science, 2017, 34(7): 1434−1441.(in Chinese) doi: 10.11829/j.issn.1001-0629.2016-0510
    [23] 黄文华. 蒙古冰草干旱胁迫下内参基因的筛选及P5CS基因定量表达分析 [D]. 呼和浩特: 内蒙古农业大学, 2014.

    HUANG W H. Selection of control gene in quantitative PCR and analysis of differential expression of P5CS gene in Agropyron mongolicum Keng under drought stress [D]. Hohhot: Inner Mongolia Agricultural University, 2014. (in Chinese).
    [24] 张燕梅, 王瑞芳, 杨子平, 等. 剑麻内参基因筛选与稳定表达分析 [J]. 热带作物学报, 2019, 40(11):2166−2173. doi: 10.3969/j.issn.1000-2561.2019.11.010

    ZHANG Y M, WANG R F, YANG Z P, et al. Screening of suitable reference genes for qRT-PCR normalization in sisal [J]. Chinese Journal of Tropical Crops, 2019, 40(11): 2166−2173.(in Chinese) doi: 10.3969/j.issn.1000-2561.2019.11.010
    [25] XIAO Z, SUN X B, LIU X Q, et al. Selection of reliable reference genes for gene expression studies on Rhododendron molle G. don [J]. Frontiers in Plant Science, 2016, 7: 1547.
    [26] HU R, QI G, KONG Y, et al. Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa [J]. BMC Plant Biology, 2010, 10(1): 145−158. doi: 10.1186/1471-2229-10-145
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出版历程
  • 收稿日期:  2020-07-29
  • 修回日期:  2020-10-04
  • 网络出版日期:  2020-11-13
  • 刊出日期:  2020-11-30

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