冬瓜实时荧光定量PCR内参基因的筛选与评价
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摘要
为筛选适合冬瓜稳定表达的内参基因,以黑皮冬瓜低温、高温、干旱胁迫处理不同时间的叶片和正常处理不同组织为试验材料,利用RT-qPCR技术,结合GeNorm、NormFinder和BestKeeper软件评价7个候选内参基因(28SrRNA、UBQ、RPⅡ、GAPDH、EF-1α、UBC21和TUA)稳定性。结果表明,7个候选内参基因在不同组织和不同胁迫处理下表达丰度及稳定性存在差异; EF-1α在低温胁迫处理下表达稳定性最好; UBQ和TUA在高温和干旱胁迫处理下表达稳定性最好; EF-1α在不同组织中表达稳定性最好。本研究结果为冬瓜内参基因的选择提供了一定的理论参考。
The present study aimed at selecting the stable reference genes to ensure the reliability and accuracy in gene expression analysis of wax gourd( Benincasa hispida Cogn.). By RT-PCR technology,combined with GeNorm,NormFinder,BestKeeper and ReFinder analysis,we investigated the expression stability of seven candidate reference genes( 28 SrRNA、UBQ、RP Ⅱ、GAPDH、EF-1α、UBC21、TUA). The results showed that,in different tissues and with different stress treatment,the expression abundance and stability of these seven reference genes were different. The expression stability of EF-1α was the best under low temperature stress.,UBQ and TUA showed the best expression stability under high temperature and drought stress,EF-1α was the best reference gene for different tissues. The results of this study can be used as a reference for the selection of internal reference genes in wax gourd.
The present study aimed at selecting the stable reference genes to ensure the reliability and accuracy in gene expression analysis of wax gourd( Benincasa hispida Cogn.). By RT-PCR technology,combined with GeNorm,NormFinder,BestKeeper and ReFinder analysis,we investigated the expression stability of seven candidate reference genes( 28 SrRNA、UBQ、RP Ⅱ、GAPDH、EF-1α、UBC21、TUA). The results showed that,in different tissues and with different stress treatment,the expression abundance and stability of these seven reference genes were different. The expression stability of EF-1α was the best under low temperature stress.,UBQ and TUA showed the best expression stability under high temperature and drought stress,EF-1α was the best reference gene for different tissues. The results of this study can be used as a reference for the selection of internal reference genes in wax gourd.
引文
[1]谢大森,何晓明,彭庆务,黄智文.冬瓜研究进展[J].华北农学报,2006(S2):166-170
[2]Huang H Y,Huang J J,Tso T K,Tsai Y C,Chang C K.Antioxidant and angiotension-converting enzyme inhibition capacities of various parts of Benincasa hispida(wax gourd)[J].Molecular Nutrition,2004,48(3):230-233
[3]张帅中,梁雪.冬瓜皮药用价值及综合利用研究进展[J].现代农业科技,2016(9):286-288
[4]Huang G L,Tan J T,Tan X C,Peng D Q.Preparation of polysaccharides from wax gourd.International journal of food sciences and nutrition[J].Food Sciences and Nutrition,2011,62(5):480-483
[5]Hooper L,Cassidy A.A review of the health care potential of bioactive compounds[J].Journal of the Science of Food andAgriculture,2006,86(12):1805-1813
[6]旷碧峰,唐锷,余席茂,肖昌华,刘小安,童龙.冬瓜搭架栽培技术[J].上海蔬菜,2016(5):40-42
[7]骆焕彬,易文红,卢文佳,李智军.特黑杂交黑皮冬瓜的特征特性及高产栽培技术[J].广东农业科学,2007(8):22-23
[8]张淑瑛.冬瓜的美容及药用价值[J].中国民间疗法,1996(4):49
[9]夏明,阮叶萍.冬瓜提取物抗小鼠胃溃疡活性研究[J].食品科学,2005(4):243-246
[10]Grover J K,Adiga G,VatsⅤ,Rathi S S.Extracts of Benincasa hispida prevent development of experimental ulcers[J].Journal of Ethnopharmacology,2001,78(213):159-164
[11]车双辉.冬瓜抗胃溃疡活性组分的筛选[D].杭州:浙江工商大学,2004
[12]邹宇晓,徐玉娟,廖森泰,张名位,张雁.冬瓜的营养价值及其综合利用研究进展[J].中国果菜,2006(5):46-47
[13]Wu C M,Liou S E,Chang Y H,Chiang W.Volatile compounds of the wax gourd(Benincasa hispida,Cogn)and a wax gourd beverage[J].Journal of Food Science,1987,52(1):132-134
[14]Pandey S,Kumar S,Mishra U,Rai A,Singh M,Rai M.Genetic diversity in Indian ash gourd(Benincasa hispida)accessions as revealed by quantitative traits and RAPD markers[J].Scientia Horticulturae,2008,118(1):80-86
[15]Jiang B,Xie D,Liu W,Peng Q,He X.De novo assembly and characterization of the transcriptome,and development of SSRmarkers in wax gourd(Benicasa hispida)[J].PLoS One,2013,8(8):e71054
[16]Jiang B,Liu W R,Xie D,Peng Q,He X,Lin Y,Liang Z.Highdensity genetic map construction and gene mapping of pericarp color in wax gourd using specific-locus amplified fragment(SLAF)sequencing[J].BMC Genomics,2015,16(1):1035
[17]Davoren P A,Mcneill R E,Lowery A J,Kerin M J,Miller N.Identification of suitable endogenous control genes for microRNAgene expression analysis in human breast cancer[J].BMC Molecular Biology,2008,9(1):76
[18]PetersⅠR,Helps C R,Hall E J,Day M J.Real-time RT-PCRconsiderations for efficient and sensitive assay design[J].Immunological Methods,2004,286(1/2):203-217
[19]Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):trends and problems[J].Molecular Endocrinology,2002,29(1):23-29
[20]Huggett J,Dheda K,Bustin S,Zumla A.Real-time RT-PCRnormalization,strategies and considerations[J].Genes and Immunity,2005,6(4):279-284
[21]朱海生,陈敏氡,温庆放,蓝新隆,李永平,王彬,张前荣,吴卫东.丝瓜18S rRNA基因克隆及其作为内参基因的应用[J].核农学报,2016,30(1):35-41
[22]Radonic A,Thuulke S,MackayⅠM,Landt O,Siegert W,Nitsche A.Guideline to reference gene selection for quantitative real-time PCR[J].Biochemical and Biophysical Research Communication,2004,313:856-862
[23]Zhang Y J,Zhu Z F,Lu R.Selection of control genes intranscription analysis of gene expression[J].Progress in Biochemistry and Biophysics,2007,34(5):546-550
[24]Artico S,Nardeli S M,Brilhante O,Grossi-De-Sa M F,AlvesFerreira M.Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data[J].BMC Plant Biology,2010,10(1):1-12
[25]庞强强,李植良,罗少波,陈日远,金庆敏,黎振兴,李德明,孙保娟,孙光闻.高温胁迫下茄qRT-PCR内参基因筛选及稳定性分析[J].园艺学报,2017,44(3):475-486
[26]张金平,何晓明,江彪,谢大森,彭庆务,王永飞,马三梅.镰刀菌酸胁迫下节瓜实时荧光定量PCR分析内参基因的选择[J].广东农业科学,2014,41(10):120-125
[27]吴建阳,何冰,杜玉洁,李伟才,魏永赞.利用geN orm、NormF inder和BestKeeper软件进行内参基因稳定性分析的方法[J].现代农业科技,2017(5):278-281
[28]Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.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
[29]Wang H L,Chen J H,Tian Q Q,Wang S,Xia X L,Yin W L.Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative realtime PCR[J].Physiologia Plantarum,2014,152(3):529-545
[30]Hong S Y,Seo P J,Yang M S,Xiang F N,Park C M.Exploring valid reference gene for gene expression studies in Brachypodium distachyon by real-time PCR[J].BMC Plant Biology,2008,8(1):112-122
[31]Lovdal T,Lillo C.Reference gene selection for quantitative realtime PCR normalization in tomato subjected to nitrogen,cold,and light stress[J].Analytical Biochemistry,2009,387(2):238-242
[32]陈杨杨,吴潇,谷超,殷豪,张绍铃.‘砀山酥梨’实时荧光定量PCR内参基因的筛选[J].中国果树,2018(1):16-22,35
[33]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,2016:15-18
[34]杜辉辉.梭梭内参基因的克隆与筛选[D].乌鲁木齐:新疆农业大学,2015:25-32
[35]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738
[36]Jain M,Nijhawan A,Tyagi A K,Khurana J P.Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J].Biochemical and Biophysical Research Communications,2006,345(2):646-651
[37]张岗,赵明明,张大为,郭顺星.铁皮石斛实时定量PCR内参基因的筛选[J].中国药学杂志,2013,48(19):1664-1668
[38]苏晓娟,樊保国,袁丽钗,崔秀娜,卢善发.实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J].植物学报,2013,48(5):507-518
[39]汪永保,余庆,李映志,李洪波,叶春海.菠萝蜜实时荧光定量PCR分析中内参基因的筛选[J].热带作物学报,2014,35(7):1374-1381
[40]Reid K E,Olsson N,Schlosser J,Peng F,Lund S T.An optimizedgrapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J].BMC Plant Biology,2006,6(1):27
[41]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
[42]肖翠,严佳文,龙桂友,戴素明,李大志,邓子牛.柑橘内参基因的稳定性评价[J].果树学报,2012,29(6):978-984
[2]Huang H Y,Huang J J,Tso T K,Tsai Y C,Chang C K.Antioxidant and angiotension-converting enzyme inhibition capacities of various parts of Benincasa hispida(wax gourd)[J].Molecular Nutrition,2004,48(3):230-233
[3]张帅中,梁雪.冬瓜皮药用价值及综合利用研究进展[J].现代农业科技,2016(9):286-288
[4]Huang G L,Tan J T,Tan X C,Peng D Q.Preparation of polysaccharides from wax gourd.International journal of food sciences and nutrition[J].Food Sciences and Nutrition,2011,62(5):480-483
[5]Hooper L,Cassidy A.A review of the health care potential of bioactive compounds[J].Journal of the Science of Food andAgriculture,2006,86(12):1805-1813
[6]旷碧峰,唐锷,余席茂,肖昌华,刘小安,童龙.冬瓜搭架栽培技术[J].上海蔬菜,2016(5):40-42
[7]骆焕彬,易文红,卢文佳,李智军.特黑杂交黑皮冬瓜的特征特性及高产栽培技术[J].广东农业科学,2007(8):22-23
[8]张淑瑛.冬瓜的美容及药用价值[J].中国民间疗法,1996(4):49
[9]夏明,阮叶萍.冬瓜提取物抗小鼠胃溃疡活性研究[J].食品科学,2005(4):243-246
[10]Grover J K,Adiga G,VatsⅤ,Rathi S S.Extracts of Benincasa hispida prevent development of experimental ulcers[J].Journal of Ethnopharmacology,2001,78(213):159-164
[11]车双辉.冬瓜抗胃溃疡活性组分的筛选[D].杭州:浙江工商大学,2004
[12]邹宇晓,徐玉娟,廖森泰,张名位,张雁.冬瓜的营养价值及其综合利用研究进展[J].中国果菜,2006(5):46-47
[13]Wu C M,Liou S E,Chang Y H,Chiang W.Volatile compounds of the wax gourd(Benincasa hispida,Cogn)and a wax gourd beverage[J].Journal of Food Science,1987,52(1):132-134
[14]Pandey S,Kumar S,Mishra U,Rai A,Singh M,Rai M.Genetic diversity in Indian ash gourd(Benincasa hispida)accessions as revealed by quantitative traits and RAPD markers[J].Scientia Horticulturae,2008,118(1):80-86
[15]Jiang B,Xie D,Liu W,Peng Q,He X.De novo assembly and characterization of the transcriptome,and development of SSRmarkers in wax gourd(Benicasa hispida)[J].PLoS One,2013,8(8):e71054
[16]Jiang B,Liu W R,Xie D,Peng Q,He X,Lin Y,Liang Z.Highdensity genetic map construction and gene mapping of pericarp color in wax gourd using specific-locus amplified fragment(SLAF)sequencing[J].BMC Genomics,2015,16(1):1035
[17]Davoren P A,Mcneill R E,Lowery A J,Kerin M J,Miller N.Identification of suitable endogenous control genes for microRNAgene expression analysis in human breast cancer[J].BMC Molecular Biology,2008,9(1):76
[18]PetersⅠR,Helps C R,Hall E J,Day M J.Real-time RT-PCRconsiderations for efficient and sensitive assay design[J].Immunological Methods,2004,286(1/2):203-217
[19]Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):trends and problems[J].Molecular Endocrinology,2002,29(1):23-29
[20]Huggett J,Dheda K,Bustin S,Zumla A.Real-time RT-PCRnormalization,strategies and considerations[J].Genes and Immunity,2005,6(4):279-284
[21]朱海生,陈敏氡,温庆放,蓝新隆,李永平,王彬,张前荣,吴卫东.丝瓜18S rRNA基因克隆及其作为内参基因的应用[J].核农学报,2016,30(1):35-41
[22]Radonic A,Thuulke S,MackayⅠM,Landt O,Siegert W,Nitsche A.Guideline to reference gene selection for quantitative real-time PCR[J].Biochemical and Biophysical Research Communication,2004,313:856-862
[23]Zhang Y J,Zhu Z F,Lu R.Selection of control genes intranscription analysis of gene expression[J].Progress in Biochemistry and Biophysics,2007,34(5):546-550
[24]Artico S,Nardeli S M,Brilhante O,Grossi-De-Sa M F,AlvesFerreira M.Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data[J].BMC Plant Biology,2010,10(1):1-12
[25]庞强强,李植良,罗少波,陈日远,金庆敏,黎振兴,李德明,孙保娟,孙光闻.高温胁迫下茄qRT-PCR内参基因筛选及稳定性分析[J].园艺学报,2017,44(3):475-486
[26]张金平,何晓明,江彪,谢大森,彭庆务,王永飞,马三梅.镰刀菌酸胁迫下节瓜实时荧光定量PCR分析内参基因的选择[J].广东农业科学,2014,41(10):120-125
[27]吴建阳,何冰,杜玉洁,李伟才,魏永赞.利用geN orm、NormF inder和BestKeeper软件进行内参基因稳定性分析的方法[J].现代农业科技,2017(5):278-281
[28]Pfaffl M W,Tichopad A,Prgomet C,Neuvians T P.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
[29]Wang H L,Chen J H,Tian Q Q,Wang S,Xia X L,Yin W L.Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative realtime PCR[J].Physiologia Plantarum,2014,152(3):529-545
[30]Hong S Y,Seo P J,Yang M S,Xiang F N,Park C M.Exploring valid reference gene for gene expression studies in Brachypodium distachyon by real-time PCR[J].BMC Plant Biology,2008,8(1):112-122
[31]Lovdal T,Lillo C.Reference gene selection for quantitative realtime PCR normalization in tomato subjected to nitrogen,cold,and light stress[J].Analytical Biochemistry,2009,387(2):238-242
[32]陈杨杨,吴潇,谷超,殷豪,张绍铃.‘砀山酥梨’实时荧光定量PCR内参基因的筛选[J].中国果树,2018(1):16-22,35
[33]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,2016:15-18
[34]杜辉辉.梭梭内参基因的克隆与筛选[D].乌鲁木齐:新疆农业大学,2015:25-32
[35]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738
[36]Jain M,Nijhawan A,Tyagi A K,Khurana J P.Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J].Biochemical and Biophysical Research Communications,2006,345(2):646-651
[37]张岗,赵明明,张大为,郭顺星.铁皮石斛实时定量PCR内参基因的筛选[J].中国药学杂志,2013,48(19):1664-1668
[38]苏晓娟,樊保国,袁丽钗,崔秀娜,卢善发.实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J].植物学报,2013,48(5):507-518
[39]汪永保,余庆,李映志,李洪波,叶春海.菠萝蜜实时荧光定量PCR分析中内参基因的筛选[J].热带作物学报,2014,35(7):1374-1381
[40]Reid K E,Olsson N,Schlosser J,Peng F,Lund S T.An optimizedgrapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J].BMC Plant Biology,2006,6(1):27
[41]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
[42]肖翠,严佳文,龙桂友,戴素明,李大志,邓子牛.柑橘内参基因的稳定性评价[J].果树学报,2012,29(6):978-984
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