李君英;张鹤;韩永峰;邵建柱;孙建设;

• 1:河北农业大学园艺学院
• 2:河北农业大学国有资产管理处

摘要(Abstract)：

【目的】筛选在组织培养条件下苹果苗中稳定表达的内参基因。【方法】应用实时荧光定量PCR技术,分析了18S r RNA,GAPDH,TUB,UBQ,ACT,EF-1α六个常用植物内参基因在苹果组培苗不同基因型、不同组织、不同继代时间的m RNA表达差异情况。供试的4个苹果基因型为‘长枝富士’‘短枝富士’‘红珍珠海棠’、砧木‘A8-11’;继代培养的不同组织为茎、叶、整株,生根培养的不同组织为根、茎、叶、整株;不同继代期间为10、20、30、40、50、60、70、80 d。【结果】经Ge Norm软件对6个内参基因的表达稳定性进行评价,ACT和EF-1α在苹果组培苗的不同组织和不同继代时间的基因表达分析中较稳定,UBQ和EF-1α在苹果组培苗不同品种中表达均稳定。【结论】组培条件下,苹果基因表达相关研究的理想内参基因为ACT、UBQ和EF-1α。

关键词(KeyWords)： 苹果;组培苗;实时荧光定量PCR;内参基因

Abstract:

Keywords:

基金项目(Foundation): 国家苹果产业技术体系专项(CARS-28)

作者(Author): 李君英;张鹤;韩永峰;邵建柱;孙建设;

Email:

DOI: 10.13925/j.cnki.gsxb.20160097

参考文献(References)：

• [1]吴然,李君英,邵建柱,孙建设,张鹤.苹果锈果类病毒实时荧光PCR检测方法的建立[J].果树学报,2015,32(1):150-155.WU Ran,LI Junying,SHAO Jianzhu,SUN Jianshe,ZHANG He.Establishment of detection method for apple scar skin viroid(ASSVd)by real-time PCR[J].Journal of Fruit Science,2015,32(1):150-155.
• [2]PODEVIN N,KRAUSS A,HENRY I,SWENNEN R,REMY S.Selection and validation of reference genes for quantitative RTPCR expression studies of the non-model crop Musa[J].Molecular Breeding,2012,30(3):1237-1252.
• [3]袁伟,万红建,杨悦俭.植物实时荧光定量PCR内参基因的特点及选择[J].植物学报,2012(4):427-436.YUAN Wei,WAN Hongjian,YANG Yuejian.The characteristic and selection of reference genes in plants for real-time fluorescence quantitative PCR[J].Chinese Bulletin of Botany,2012(4):427-436.
• [4]HAN B,YANG Z,SAMMA M K,WANG R,SHEN W.Systematic validation of candidate reference genes for q RT-PCR normalization under iron deficiency in Arabidopsis[J].Biometals,2013,26(3):403-413.
• [5]张玉芳,赵丽娟,曾幼玲.基因表达研究中内参基因的选择与应用[J].植物生理学报,2014(8):1119-1125.ZHANG Yufang,ZHAO Lijuan,ZENG Youling.Selection and application of reference genes for gene expression studies[J].Plant Physiology Journal,2014(8):1119-1125.
• [6]赵玉山.我国苹果产业发展浅析[J].科学种养,2014(9):5-7.ZHAO Yushan.The brief analysis of apple industry development in our country[J].Kexue Zhongyang,2014(9):5-7.
• [7]李保华,王彩霞,董向丽.我国苹果主要病害研究进展与病害防治中的问题[J].植物保护,2013,23(5):46-54.LI Baohua,WANG Caixia,DONG Xiangli.The research progress of the main apple diseases and the problems in preventing diseases in China[J].Plant Protection,2013,23(5):46-54.
• [8]陈红霞.我国苹果主产区潜隐性病毒侵染特征研究[D].保定:河北农业大学,2012.CHEN Hongxia.Study on infection characteristics of apple latent Virus in China’s main producing areas[D].Baoding:Agricultural University of Hebei,2012.
• [9]姜中武,宋来庆,梁成林.苹果病毒病与无病毒苗木建园[J].烟台果树,2014(1):27-30.JIANG Zhongwu,SONG Laiqing,LIANG Chenglin.Apple virus diseases and the establishment of apple orchards with virus-free seeding[J].Yantai Fruits,2014(1):27-30.
• [10]KUMAR K,MUTHAMILARASAN M,PRASAD M.Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet(Setaria italica L.)subjected to abiotic stress conditions[J].Plant Cell Tissue and Organ Culture,2013,115(1):13-22.
• [11]JACOB P B,ROBIN V B,RISCHERC H,GOOSSENS A.Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus[J].Plant Physiology and Biochemistry,2014,83C:20-25.
• [12]ZHUANG H,FU Y,HE W,WANG L,WEI Y.Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments[J].Frontiers in Plant Science,2015,6(475):475.
• [13]CHEN J,LI X,WANG D,LI L,ZHOU H.Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri[J].Scientia Horticulturae,2015,190:43-56.
• [14]周兰,张利义,张彩霞,康国栋,田义,丛佩华.苹果实时荧光定量PCR分析中内参基因的筛选[J].果树学报,2012,29(6):965-970.ZHOU Lan,ZHANG Liyi,ZHANG Caixia,KANG Guodong,TIAN Yi,CONG Peihua.Screening of reference genes for realtime fluorescence quantitative PCR in apple(Malus×domestica)[J].Journal of Fruit Science,2012,29(6):965-970.
• [15]樊连梅,王超,刘更森,原永兵.苹果着色期实时定量PCR内参基因的筛选和验证[J].植物生理学报,2014(12):1903-1911.FAN Lianmei,WANG Chao,LIU Gengsen,YUAN Yongbing.Screening and validation of reference genes for real-time fluorescence quantitative PCR during coloring period in apple(Malus domestica)[J].Plant Physiology Journal,2014(12):1903-1911.
• [16]YE X,ZHANG F,TAO Y,SONG S,FANG J.Reference gene selection for quantitative real-time PCR normalization in different cherry genotypes,developmental stages and organs[J].Scientia Horticulturae,2015,181:182-188.
• [17]XU Y,LI H,LI X G,LIN J,YANG Q S,CHANG Y H.Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear[J].Acta Physiologiae Plantarum,2015,37:40.
• [18]CHEN L,ZHONG H Y,KUANG J F,LU W J,CHEN J Y.Validation of reference genes for RT-q PCR studies of gene expression in banana fruit under different experimental conditions[J].Planta,2011,234(2):377-390.
• [19]GALLI V,BOROWSKI JM,PERIN E C,MESSIAS R S,ROMBALDI C V.Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses[J].Gene,2015(2):205-214.
• [20]WU J,SU S,FU L,ZHANG Y,CHAI L,YI H.Selection of reliable reference genes for gene expression studies using quantitative real-time PCR in navel orange fruit development and pummelo floral organs[J].Scientia Horticulturae,2014,176:180-188.
• [21]WAN H,YUAN W,RUAN M,YE Q,YANG Y.Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper(Capsicum annuum L.)[J].Biochemical and Biophysical Research Communications,2011,416(1-2):24-30.
• [22]ZHOU X,LIU J,ZHUANG Y.Selection of appropriate reference genes in eggplant for quantitative gene expression studies under different experimental conditions[J].Scientia Horticulturae,2014,176:200-207.
• [23]XU Y,ZHU X,GONG Y,LIU L.Evaluation of reference genes for gene expression studies in radish(Raphanus sativus L.)using quantitative real-time PCR[J].Biochemical and Biophysical Research Communications,2012,424(3):398-403.
• [24]WANG T,LU J X,ZHANG Q X.Selection of suitable reference genes for mi RNA expression normalization by q RT-PCR during flower development and different genotypes of Prunus mume[J].Scientia Horticulturae,2014,169:130-137.
• [25]LUO H,LUO K,LI E X,GUAN B,XIONG D J,YANG B.Evaluation of candidate reference genes for gene expression studies in Cymbidium kanran[J].Scientia Horticulturae,2014,167:43-48.