红梨实时荧光定量PCR内参基因的筛选
|
摘要
红梨(Pyrus pyrifolia)果皮花青苷的合成积累是影响红梨果实着色的关键因素,其生物合成由各步酶促反应基因调控,筛选出稳定可靠的内参基因对于研究红梨果实着色关键基因及其调控机制具有重要意义。本研究旨在筛选红梨的不同品种(早白蜜,中熟32,云红梨1号),在不同生长发育时期(幼果期,缓慢生长期,快速生长期,成熟期)和不同遮光处理(自然光照,部分遮光及完全遮光)条件下均稳定表达的内参基因,以用于qRT-PCR分析。选取红梨果皮为实验材料,以梨树(Pyrus communis)中常见的6个管家基因—β-肌动蛋白基因(β-actin, ACT)、18S核糖体RNA基因(18S ribosomal RNA, 18S rRNA)、甘油醛三磷酸脱氢酶基因(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)、组蛋白基因(histone, His)、转录延伸因子基因(elongation factor 1α, EF-1α)及α微管蛋白基因(α-tubulin, TUB)作为候选内参基因,分别进行q RT-PCR扩增效率、内参基因稳定性参数及不同样品中相对表达量分析。以BIO-RAD CFX Manager v2.0软件对稳定性参数指标—循环阈值(cycle threshold, Ct)、平均表达系数(M)和变异系数(variation coefficient, CV)进行分析和综合对比,结果显示,不同候选内参基因的表达稳定性存在差异,其中EF-1α和His在红梨不同品种、不同生长发育时期及不同遮光处理条件下均稳定表达;对于不同样本中内参基因相对表达量的分析显示,EF-1α和His在不同样本中的表达量相对稳定。上述结果提示,EF-1α和His可作为红梨果皮组织qRTPCR分析的内参基因,为后续开展红梨果实着色相关基因的表达分析提供了校正和标准化基因选择。
Synthesis and accumulation of cyanine nucleoside in Red pear(Pyrus pyrifolia) peel are key factors influencing red pear fruit coloring and biosynthesis regulated by enzymatic reaction genes, screening of stable and reliable reference genes has important significance to research the key genes and their regulatory mechanism in red pear fruit coloring. In order to select reference genes for qRT-PCR analysis in different growth period(young fruit period, slow growth period, fast growth period, mature period) and different shading conditions(natural light, partial shading, complete shading) in different varieties(Zaobaimi,Zhongshu32, Yunhongli No.1) of red pear fruit, 6 common house-keeping genes were chosen as candidate forinternal gene which included β-actin(ACT), 18 S ribosomal RNA(18 S rRNA), glyceraldehyde-3-phosphate dehydrogenase gene(GAPDH), histone(His), elongation factor 1α(EF-1α), and α-tubulin(TUB). The qRTPCR amplification efficiency, the stability parameters and the relative expression in different samples of internal reference genes were analyzed. The BIO-RAD CFX Manager v2.0 software was used to analyze and compare the stability parameter index such as cycle threshold(Ct), average expression coefficient(M) and variation coefficient(CV). The results showed different expression stability in different candidate genes, and EF-1α and His showed stable expression trend in different samples from different varieties, growth stages, and processing conditions. Analysis of relative expression of reference genes in different samples showed that the expression of EF-1α and His in different samples were relatively stable. As a result, EF-1α and His could be used as reference gene for qRT-PCR analysis in red pear peel tissues which might standardize genetic selection for relative quantitation about coloring related gene expression analysis in red pear.
Synthesis and accumulation of cyanine nucleoside in Red pear(Pyrus pyrifolia) peel are key factors influencing red pear fruit coloring and biosynthesis regulated by enzymatic reaction genes, screening of stable and reliable reference genes has important significance to research the key genes and their regulatory mechanism in red pear fruit coloring. In order to select reference genes for qRT-PCR analysis in different growth period(young fruit period, slow growth period, fast growth period, mature period) and different shading conditions(natural light, partial shading, complete shading) in different varieties(Zaobaimi,Zhongshu32, Yunhongli No.1) of red pear fruit, 6 common house-keeping genes were chosen as candidate forinternal gene which included β-actin(ACT), 18 S ribosomal RNA(18 S rRNA), glyceraldehyde-3-phosphate dehydrogenase gene(GAPDH), histone(His), elongation factor 1α(EF-1α), and α-tubulin(TUB). The qRTPCR amplification efficiency, the stability parameters and the relative expression in different samples of internal reference genes were analyzed. The BIO-RAD CFX Manager v2.0 software was used to analyze and compare the stability parameter index such as cycle threshold(Ct), average expression coefficient(M) and variation coefficient(CV). The results showed different expression stability in different candidate genes, and EF-1α and His showed stable expression trend in different samples from different varieties, growth stages, and processing conditions. Analysis of relative expression of reference genes in different samples showed that the expression of EF-1α and His in different samples were relatively stable. As a result, EF-1α and His could be used as reference gene for qRT-PCR analysis in red pear peel tissues which might standardize genetic selection for relative quantitation about coloring related gene expression analysis in red pear.
引文
陈杨杨,吴潇,谷超,等.2018.‘砀山酥梨’实时荧光定量PCR内参基因的筛选[J].中国果树,(1):16-22,35(Chen Y Y,Wu X,Gu C,et al.2018.Selection of reference genes in qRT-PCR of pear‘Dangshansuli’[J].China Fruit,(1):16-22,35.)
丁锐.2004.国外花色素苷的研究现状与进展[J].汉中师范学院学报,22(2):73-78.(Ding R.2004.The foreign research status and progress of anthocyanin[J].Journal of Hanzhong Teachers College,22(2):73-78.)
古丽娜尔·夏依马尔旦.2007.和田玫瑰花红色素的提取及性质研究[J].新疆师范大学学报,26(2):84-88(Gulnar Xiayimardan.2007.Refinement of the pigmen of hetian rose andstudy of its nature[J].Journal of Xinjiang Normal University(Natural Sciences Edition),26(2):84-88.)
洪阳,尹俊梅,黄少华,等.2015.红掌细菌性叶疫病胁迫下实时荧光定量PCR(qRT-PCR)内参基因的筛选[J].农业生物技术学报,23(9):1178-1187.(Hong Y,Yin J MHuang S H,et al.2015.Reference gene selection for quantitative real-time PCR(qRT-PCR)in Anthurium(Anthurium andraeanum Linden)tissues under bacteria blight stress[J].Journal of Agricultural Biotechnology23(9):1178-1187.)
候夫云,王庆美,李爱贤,等.2009.植物花青素合成酶的研究进展[J].中国农学通报,25(21):188-190.(Hou F YWang Q M,Li A X,et al.2009.Study progress on anthocyanidin synthase of plants[J].Chinese Agricultura Science Bulletin,25(21):188-190.)
侯维海,孙鹏,陈全家,等.2011.地黄实时定量PCR内参基因的筛选[J].中国农学通报,27(17):76-82.(Hou W HSun P,Chen Q J,et al.2011.Selection of the Reference genes for gene expression studies in Rehmannia glutinosaby real-time quantitative PCR[J].Chinese Agricultural Science Bulletin,27(17):76-82.)
胡瑞波,范成明,傅永福.2009.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报,11(6):30-36(Hu R B,Fan C M,Fu Y F.2009.Reference gene selection in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Journal of Agricultural Science and Technology,11(6):30-36.)
刘金泊,欧静,姚富姣,等.2014.磷化氢诱导下赤拟谷盗实时定量PCR内参基因的筛选[J].农业生物技术学报22(2):257-264.(Liu J B,Ou J,Yao F J,et al.2014.Identification of appropriate reference genes for gene expression studies by quantitative real-time PCR in Tribolium castaneum after exposure to phosphine[J].Journal of Agricultural Biotechnology,22(2):257-264.)
李雪,潘学军,张文娥,等.2017.核桃内参基因实时荧光定量PCR表达稳定性评价[J].植物生理学报,53(9):1795-1802.(Li X,Pan X J,Zhang W E,et al.Evaluation of real-time PCR expression stability of internal reference gene in walnut[J].Plant Physiology Journal,53(9):1795-1802.)
卢钰,董现义,杜景平,等.2004.花色苷研究进展[J].山东农业大学学报,35(2):315-320.(Lu Y,Dong X Y,Du J P,et al.2004.Research progress on anthocyanins[J].Journal of Shangdong Agricultural University(Natural Science),35(2):315-320.)
王梨寰,潘永娟,杨莉,等.2013.‘琯溪蜜柚’荧光定量PCR内参基因的筛选[J].果树学报,30(1):48-54.(Wang LH,Pan Y J,Yang L,et al.2013.Validation of internal reference genes for q RT-PCR normalization in‘Guanxi Sweet Pummelo’(Citrus grandis)[J].Journal of Fruit Science,30(1):48-54.)
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[J].农业生物技术学报,20(5):521-528.(Wang Y J,Dong L,Zhang C,et al.2012.Reference gene selection for real-time quantitative PCR normalization in tree peony(Paeonia suffruticosa Andr).[J].Journal of Agricultural Biotechnology,20(5):521-528.)
魏毅东,陈玉,郭海萍,等.2013.水稻缺素胁迫下实时荧光定量RT-PCR的内参基因的选择[J].农业生物技术学报,21(11):1302-1312.(Wei Y D,Chen Y,Guo H P,et al.2013.Selection of reference genes for real-time quantitative RT-PCR in rice(Oryza sativa L.ssp.japonica)under nutrient deficiency[J].Journal of Agricultural Biotechnology,21(11):1302-1312.)
徐丽华,刘春雷,常玉梅,等.2011.双标准曲线相对定量PCR试验原理与方法[J].生物技术通报,(01):70-75.(Xu LH,Liu C L,Chang Y M,et al.2011.Theory and method of double-standard curves method of relative quantification PCR[J].Biotechnology Bulletin,(01):70-75.)
尹冬梅,赵振宇,郭凤根,等.2017.岩白菜实时荧光定量PCR分析的内参基因的筛选[J].基因组学与应用生物学,36(10):4256-4262.(Ying D M,Zhao Z Y,Guo F G,et al.2017.Screening of reference genes for real-time quantitative PCR in Bergenia purpurascens[J].Genomics and Applied Biology,36(10):4256-4262.)
袁伟,万红建,阳悦俭.2012.植物实时荧光定量PCR内参基因的特点及选择[J].植物学报,47(4):427-436.(Yuan W,Wan H J,Yang Y J.2012.Characterization and selection of reference gene in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Chinese Bulletin of Botany,47(4):427-436.)
查倩,奚晓军,蒋爱丽,等.2016.葡萄实时定量PCR中稳定内参基因的筛选[J].果树学报,33(3):268-274.(Zha Q,Xi X J,Jiang A L,et al.2016.Identification of the appropriate reference gene through using a realtime quantitative PCR in grapes[J].Journal of Fruit Science,33(3)268-274.)
周兰,张利义,张彩霞,等.2012.苹果实时荧光定量PCR分析中内参基因的筛选[J].果树学报,29(6):965-970(Zhou L,Zhang L Y,Zhang C X,et al.Screening of reference genes for real-time fluorescence quantitative PCR in apple(Malus domestica)[J].Journal of Frui Scrence,29(6):965-970.)
Argyropoulos D,Psallida C,Spyropoulos C G.2006.Generic normalization method for real-time PCR.Application for the analysis of the mannanase gene expressed in germinating tomato seed[J].The FEBS Journal,273(4)770-777.
Bustin S A.2002.Quantification of mRNA using real-time reverse transcription PCR RT-PCR:Trends and problems[J].Journal of Molecular Endocrinol,29(1):23-29.
Chen Y Q,Li X Y,Wang D Q,et al.2015.Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri[J].Scientia Horticulturae,190(16):43-56.
Freeman W M,Walker S J,Vrana K E.1999.Quantitative RT-PCR:Pitfalls and potential[J].BioTechniques,26(1)112-122,124-125.
David W.2000.Regulation of flower pigmentation and growth:Multiplesignaling pathways control anthocyanin synthesis in expanding petals[J].Physiologia Plantarum,110(2):152-157.
Imai T,Ubi B E,Saito T,et al.2014.Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions[J].PLoSOne,9(1):e86492.
Kim B R,Nam H Y,Kim S U,et al.2003.Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J].Biotechnology Letters,25(21):1869-1872.
Laurent G,Mélanie M,Stéphanie G,et al.2008.The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcriptionpolymerase chain reaction(RT-PCR)analysis in plants[J].Plant Biotechnology Journal,6(6):609-618.
Suzuki T,Higgins P J,Crawford D.2000.Control selection for RNA quantitation[J].Biotechniques,29(2):332-337.
Thellin O,Zorzi W,Lakaye B,et al.1999.Housekeeping genes as internal standards:Use and limits[J].Jouranl of Biotechnolgy,75(2-3):291-295.
Xu Y Y,Li H,Li X G,et al.2015.Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear[J].Acta Physiologiae Plantarum,37(2):1-16.
丁锐.2004.国外花色素苷的研究现状与进展[J].汉中师范学院学报,22(2):73-78.(Ding R.2004.The foreign research status and progress of anthocyanin[J].Journal of Hanzhong Teachers College,22(2):73-78.)
古丽娜尔·夏依马尔旦.2007.和田玫瑰花红色素的提取及性质研究[J].新疆师范大学学报,26(2):84-88(Gulnar Xiayimardan.2007.Refinement of the pigmen of hetian rose andstudy of its nature[J].Journal of Xinjiang Normal University(Natural Sciences Edition),26(2):84-88.)
洪阳,尹俊梅,黄少华,等.2015.红掌细菌性叶疫病胁迫下实时荧光定量PCR(qRT-PCR)内参基因的筛选[J].农业生物技术学报,23(9):1178-1187.(Hong Y,Yin J MHuang S H,et al.2015.Reference gene selection for quantitative real-time PCR(qRT-PCR)in Anthurium(Anthurium andraeanum Linden)tissues under bacteria blight stress[J].Journal of Agricultural Biotechnology23(9):1178-1187.)
候夫云,王庆美,李爱贤,等.2009.植物花青素合成酶的研究进展[J].中国农学通报,25(21):188-190.(Hou F YWang Q M,Li A X,et al.2009.Study progress on anthocyanidin synthase of plants[J].Chinese Agricultura Science Bulletin,25(21):188-190.)
侯维海,孙鹏,陈全家,等.2011.地黄实时定量PCR内参基因的筛选[J].中国农学通报,27(17):76-82.(Hou W HSun P,Chen Q J,et al.2011.Selection of the Reference genes for gene expression studies in Rehmannia glutinosaby real-time quantitative PCR[J].Chinese Agricultural Science Bulletin,27(17):76-82.)
胡瑞波,范成明,傅永福.2009.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报,11(6):30-36(Hu R B,Fan C M,Fu Y F.2009.Reference gene selection in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Journal of Agricultural Science and Technology,11(6):30-36.)
刘金泊,欧静,姚富姣,等.2014.磷化氢诱导下赤拟谷盗实时定量PCR内参基因的筛选[J].农业生物技术学报22(2):257-264.(Liu J B,Ou J,Yao F J,et al.2014.Identification of appropriate reference genes for gene expression studies by quantitative real-time PCR in Tribolium castaneum after exposure to phosphine[J].Journal of Agricultural Biotechnology,22(2):257-264.)
李雪,潘学军,张文娥,等.2017.核桃内参基因实时荧光定量PCR表达稳定性评价[J].植物生理学报,53(9):1795-1802.(Li X,Pan X J,Zhang W E,et al.Evaluation of real-time PCR expression stability of internal reference gene in walnut[J].Plant Physiology Journal,53(9):1795-1802.)
卢钰,董现义,杜景平,等.2004.花色苷研究进展[J].山东农业大学学报,35(2):315-320.(Lu Y,Dong X Y,Du J P,et al.2004.Research progress on anthocyanins[J].Journal of Shangdong Agricultural University(Natural Science),35(2):315-320.)
王梨寰,潘永娟,杨莉,等.2013.‘琯溪蜜柚’荧光定量PCR内参基因的筛选[J].果树学报,30(1):48-54.(Wang LH,Pan Y J,Yang L,et al.2013.Validation of internal reference genes for q RT-PCR normalization in‘Guanxi Sweet Pummelo’(Citrus grandis)[J].Journal of Fruit Science,30(1):48-54.)
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[J].农业生物技术学报,20(5):521-528.(Wang Y J,Dong L,Zhang C,et al.2012.Reference gene selection for real-time quantitative PCR normalization in tree peony(Paeonia suffruticosa Andr).[J].Journal of Agricultural Biotechnology,20(5):521-528.)
魏毅东,陈玉,郭海萍,等.2013.水稻缺素胁迫下实时荧光定量RT-PCR的内参基因的选择[J].农业生物技术学报,21(11):1302-1312.(Wei Y D,Chen Y,Guo H P,et al.2013.Selection of reference genes for real-time quantitative RT-PCR in rice(Oryza sativa L.ssp.japonica)under nutrient deficiency[J].Journal of Agricultural Biotechnology,21(11):1302-1312.)
徐丽华,刘春雷,常玉梅,等.2011.双标准曲线相对定量PCR试验原理与方法[J].生物技术通报,(01):70-75.(Xu LH,Liu C L,Chang Y M,et al.2011.Theory and method of double-standard curves method of relative quantification PCR[J].Biotechnology Bulletin,(01):70-75.)
尹冬梅,赵振宇,郭凤根,等.2017.岩白菜实时荧光定量PCR分析的内参基因的筛选[J].基因组学与应用生物学,36(10):4256-4262.(Ying D M,Zhao Z Y,Guo F G,et al.2017.Screening of reference genes for real-time quantitative PCR in Bergenia purpurascens[J].Genomics and Applied Biology,36(10):4256-4262.)
袁伟,万红建,阳悦俭.2012.植物实时荧光定量PCR内参基因的特点及选择[J].植物学报,47(4):427-436.(Yuan W,Wan H J,Yang Y J.2012.Characterization and selection of reference gene in plant real-time quantitative reverse transcription PCR(qRT-PCR)[J].Chinese Bulletin of Botany,47(4):427-436.)
查倩,奚晓军,蒋爱丽,等.2016.葡萄实时定量PCR中稳定内参基因的筛选[J].果树学报,33(3):268-274.(Zha Q,Xi X J,Jiang A L,et al.2016.Identification of the appropriate reference gene through using a realtime quantitative PCR in grapes[J].Journal of Fruit Science,33(3)268-274.)
周兰,张利义,张彩霞,等.2012.苹果实时荧光定量PCR分析中内参基因的筛选[J].果树学报,29(6):965-970(Zhou L,Zhang L Y,Zhang C X,et al.Screening of reference genes for real-time fluorescence quantitative PCR in apple(Malus domestica)[J].Journal of Frui Scrence,29(6):965-970.)
Argyropoulos D,Psallida C,Spyropoulos C G.2006.Generic normalization method for real-time PCR.Application for the analysis of the mannanase gene expressed in germinating tomato seed[J].The FEBS Journal,273(4)770-777.
Bustin S A.2002.Quantification of mRNA using real-time reverse transcription PCR RT-PCR:Trends and problems[J].Journal of Molecular Endocrinol,29(1):23-29.
Chen Y Q,Li X Y,Wang D Q,et al.2015.Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri[J].Scientia Horticulturae,190(16):43-56.
Freeman W M,Walker S J,Vrana K E.1999.Quantitative RT-PCR:Pitfalls and potential[J].BioTechniques,26(1)112-122,124-125.
David W.2000.Regulation of flower pigmentation and growth:Multiplesignaling pathways control anthocyanin synthesis in expanding petals[J].Physiologia Plantarum,110(2):152-157.
Imai T,Ubi B E,Saito T,et al.2014.Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions[J].PLoSOne,9(1):e86492.
Kim B R,Nam H Y,Kim S U,et al.2003.Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice[J].Biotechnology Letters,25(21):1869-1872.
Laurent G,Mélanie M,Stéphanie G,et al.2008.The lack of a systematic validation of reference genes:A serious pitfall undervalued in reverse transcriptionpolymerase chain reaction(RT-PCR)analysis in plants[J].Plant Biotechnology Journal,6(6):609-618.
Suzuki T,Higgins P J,Crawford D.2000.Control selection for RNA quantitation[J].Biotechniques,29(2):332-337.
Thellin O,Zorzi W,Lakaye B,et al.1999.Housekeeping genes as internal standards:Use and limits[J].Jouranl of Biotechnolgy,75(2-3):291-295.
Xu Y Y,Li H,Li X G,et al.2015.Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear[J].Acta Physiologiae Plantarum,37(2):1-16.