花椰菜BobACT基因的克隆及其作为内参基因的研究
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摘要
实时荧光定量PCR技术是探索植物基因功能和调节机理的有效手段。选择合适的内参基因是获得实时荧光定量PCR准确性数据的必备条件。ACT基因高度保守且表达稳定,常作为内参基因被广泛应用。为了获得花椰菜ACT基因,以转录组测序和RT-PCR方法为手段克隆得到花椰菜肌动蛋白基因Actin。该基因等电点为5.395,理论分子量为41.77 kD;其cDNA开放阅读框长1134 bp,编码氨基酸377个,GenBank登录号为MG598643。Wolf Psort分析发现,BobActin蛋白亚细胞定位于细胞质基质中。Motif Scan分析显示,BobActin蛋白质的氨基酸序列4～377位为Actin保守结构域。进化分析表明,同源序列基因编码的蛋白质与同为十字花科的甘蓝、芜菁和油菜同源蛋白的相似性达到90%以上,具有高度的保守性。在此基础上,设计了1对荧光定量PCR引物,分析显示,该引物具有较高的特异性和扩增效率,在花椰菜根、茎、花、花球、叶片等不同组织和低温、高温、盐处理、干旱处理、ABA处理等胁迫处理下均能稳定表达,适合在花椰菜基因表达研究中作为内参基因,为开展花椰菜重要功能基因的挖掘、表达模式以及调控机理的研究提供参考。花椰菜在内参基因方面的研究还处于初步阶段,今后可继续克隆其他内参基因,丰富花椰菜的内参基因库,从而进一步提高花椰菜基因表达分析研究的稳定性、重复性和准确性。
Real-time fluorescence quantitative PCR is an effective method to quantify the transcriptional profile of target genes.Use of proximal gene as internal reference is essential when performing qRT-PCR experiments.The Actin(ACT)gene is highly conserved cross species and expressed stably and is often used as an internal control.In order to obtain the ACT gene of cauli?ower,the ACT gene of cauli?ower was cloned by RT-PCR method(GenBank ID:MG598643).The open reading frame(ORF)is 1134 bp,encoding 377 amino acids with a predicted molecular weight of 41.77 kD and a hypothetical isoelectric point of 5.395.Wolf Psort analysis indicated that BobActin protein was located in the cytoplasmic matrix,and Motif Scan analysis showed that BobActin protein had the conserved actin at position of 4-377 sites.BobActin shared 90% identity with the homologous proteins from genus Brassicacea,such as Brassica oleracea L.var.oleracea,Brassica rapa L.and Brassica napus L..A pair of qRT-PCR primers was designed from the BobActin gene sequence,and this combination showed high speci?city and ampli?cation ef?ciency.qRT-PCR analysis indicated that the BobActin gene was stably expressed in tissues of cauli?ower,including root,stem,?ower ball,leaf,even under various stress treatments(low temperature,high temperature,salt,drought and ABA).Thus,this gene might serve as an internal reference being suitable for gene expression in cauli?ower.
Real-time fluorescence quantitative PCR is an effective method to quantify the transcriptional profile of target genes.Use of proximal gene as internal reference is essential when performing qRT-PCR experiments.The Actin(ACT)gene is highly conserved cross species and expressed stably and is often used as an internal control.In order to obtain the ACT gene of cauli?ower,the ACT gene of cauli?ower was cloned by RT-PCR method(GenBank ID:MG598643).The open reading frame(ORF)is 1134 bp,encoding 377 amino acids with a predicted molecular weight of 41.77 kD and a hypothetical isoelectric point of 5.395.Wolf Psort analysis indicated that BobActin protein was located in the cytoplasmic matrix,and Motif Scan analysis showed that BobActin protein had the conserved actin at position of 4-377 sites.BobActin shared 90% identity with the homologous proteins from genus Brassicacea,such as Brassica oleracea L.var.oleracea,Brassica rapa L.and Brassica napus L..A pair of qRT-PCR primers was designed from the BobActin gene sequence,and this combination showed high speci?city and ampli?cation ef?ciency.qRT-PCR analysis indicated that the BobActin gene was stably expressed in tissues of cauli?ower,including root,stem,?ower ball,leaf,even under various stress treatments(low temperature,high temperature,salt,drought and ABA).Thus,this gene might serve as an internal reference being suitable for gene expression in cauli?ower.
引文
[1]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·核农学报,2016, 30(1):35-41Zhu H S, Chen M D, Wen Q F, Lan X L, Li Y P, Wang B,Zhang Q R, Wu W D-Cloning of 185 rRNA gene from Luff a cylindrical and its application as an internal standard.Journal of Nuclear Agricultural Sciences, 2016, 30(1):35-41
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[3]蒋晓梅,张新全,严海东,张瑜,杨盛婷,黄琳凯·柳枝稷根组织实时定量PCR分析中内参基因的选择·农业生物技术学报,2014, 22(1):55-63Jiao X M, Zhang X Q, Yan H D, Zhang Y, Yang S T, Huang L K-Reference gene selection for real-time quantitative PCR normalization in switchgrass(Panicum virgatum L.)root tissue.Journal of Agricultural Biotechnology, 2014, 22(1):55-63
[4]Wan H J,Zhao Z G,Qian C T,Chen J F.Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber.Analytical Biochemistry, 2009, 399:257-261
[5]Gutierrez L,Mauriat M,Pelloux J,Bellini C,Wuytswinkel O.Towards a systematic validation of references in real-time RTPCR.The Plant Cell, 2008,20:1734-1735
[6]Goulao L F,Fortunato A S,Ramalho J C.Selection of reference genes for normalizing quantitative real-time PCR gene expression data with multiple variables in Coffea spp-Plant Molecular Biology Reporter, 2012,30:741-759
[7] Zhu X Y, Li X P, Chen W X, Chen J Y, Lu W J, Chen L, Fu D W.Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.PLoS One,2012,7(8):e44405
[8]杨丹,李清,王贵禧,马庆华,朱利泉.平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立.中国农业科学,2017,50(12):2399-2410Yang D, Li Q, Wang Q X, Ma Q H, Zhu L Q.Reference genes selection and system establishment for real-time qPCR analysis in Ping'ou hybrid hazelnut(C.heterophylla Fisch. x C.avellana L.)-Scientia Agricultura Sinica, 2017,50(12):2399-2410
[9]Karuppaiya P,Yan X X,Liao W,Wu J,Chen F,TangL-Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas-A biodiesel plant.PLoS One,2017, 12(2):e0172460
[10] Zhang L C, Liu L, Cheng P, Shen H G, Rong B, Liu W, Yu G H.Identification and validation of reference genes for RT-qPCR analysis in banana(Musa spp.)under Fusarium wilt resistance induction conditions. Journal of Phytopathology,2017,165(11-12):746-754
[11]Fu Y,Duan X,Tang C,Li X,Voegele R T,Wang X,Wei G,Kang Z.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici.The Plant Journal, 2014, 78(1):16-30
[12] Dominguez R, Holmes K C. Actin structure and function.Annual review of biophysics, 2011,40(1):69
[13]崔力文,郑婷,张克坤,张川,上官凌飞,房经贵·葡萄Actin基因家族的鉴定及进化和表达分析·植物资源与环境学报,2017,26(3):1-10Cui L W, Zheng T, Zhang K K, Zhang C, Shangguan L F,Fang J G-Identification, evolution and expression analyses of Actin gene family of Vitis vinifera.Journal of Plant Resources and Environment, 2017,26(3):1-10
[14]李文萍,林俊城,黄科·全球花椰菜生产与贸易现状分析·中国蔬菜,2014(9):5-10Li W P,Lin J C,Huang K.Analysis about present status of global cauliflower production and its trade-China Vegetables,2014,(9):5-10
[15]顾宏辉,金昌林,赵振卿,盛小光,虞慧芳,王建升·我国松花菜产业现状及前景分析·中国蔬菜,2012(23):1-5Gu H H,Jin C L,Zhao Z Q,Sheng X G, Yu H F, Wang J S.Status quo and prospect in food industry in China-China Vegetables,2012(23):1-5
[16]丁云花,何洪巨,赵学志,王文琪,宋曙辉·不同类型花椰菜主要营养品质分析·中国蔬菜,2016(4):58-63Ding Y H, He H J, Zhao X Z, Wang W Q, Song S H.Analysis of major nutritional quality of different types of cauliflower varieties-China Vegetables, 2016(4):58-63
[17] Zhu H S, Liu J T, Wen Q F, Chen M D, Wang B, Zhang Q R,Xue ZZ, De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica'Fusi-3'fruits.PLoS One, 2017, 12(11):e0187117
[18]刘建汀,朱海生,温庆放,王彬,张前荣,陈敏氡,林珲,薛珠政.丝瓜LcWRKY21转录因子基因的克隆与表达分析·中国细胞生物学学报,2017, 39(10):1268-1278Liu J T, Zhu H S, Wen Q F, Wang B, Zhang Q R, Chen M D,Lin H, Xue Z Z.Cloning and expression analysis of transcription factor LcWRKY21 gene from Luffa cylindrical-Chinese Journal of Cell Biology, 2017, 39(10):1268-1278
[19]温庆放,刘建汀,朱海生,陈敏氡,王彬,张前荣·丝瓜过氧化氢酶基因(CAT1)的克隆及表达分析·园艺学报,2016,43(10):2039-2048Wen Q F, Liu J T, Zhu H S, Chen M D, Wang B, Zhang Q R Cloning and expression analysis of catalase CAT1 gene from Luffa cylindrical-Acta Horticulturae Sinica, 2016,43(10):2039-2048
[20]Mascia T, Santovito E,Gallitelli D,Cillo F.Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants-MolecularPlant Pathology, 2010, 11(6):805-816
[21]秦剑英,刘灶长,孔德艳,周立国,罗利军·适合于miRNA的荧光定量PCR优化体系的建立及其标准·植物遗传资源学报,2012, 13(3):435-442Qin J Y, Liu Z Z, Kong D Y, Zhou L G, Luo L J-Establishment and Criteria of Optimal Reaction System of RT-qPCR Suitable for miRNA.Journal of Plant Genetic Resources, 2012,13(3):435-442
[22]崔艳伟,李文龙,常文锁,李喜焕,张彩英·大豆异黄酮合成途径相关基因差异表达分析·植物遗传资源学报,2016, 17(4):719-725Cui Y W, Li W L, Chang W S, Li X H, Zhang C X-Differential Expression Analysis of Genes Associated with Isoflavone Synthesis in Soybean(Glycine max Merr.).Journal of Plant Genetic Resources, 2016, 17(4):719-725-
[23]刘建汀,朱海生,王彬,李永平,陈敏氡,张前荣,温庆放·西葫芦CpActin内参基因的分离及其作为内参基因的初步应用·植物遗传资源学报,2019, 20(1):188-198Liu J T,Zhu H S,Wang B,Li Y P,Chen M D,Zhang Q L,Wen Q F-Isolation of CpActin Gene and Study on This Gene as Reference Gene in cucurbita pepo. Journal of Plant Genetic Resources, 2019,20(1):188-198
[24] Udvardi M K, Czechowski T, Scheible W R-Eleven golden rules of quantitative RT-PCR-Plant Cell, 2008,20(7):1736-1737
[25] Wrzesi n ska B, Kierzek R, Obrepalska. Steplowska A.Evaluation of six commonly used reference genes for gene expression studies in herbicide-resistant Avena fatua biotypesWeed Research, 2016, 56(4):284-292
[26] Chen L, Zhong H Y, Kuang J F, Li J G, Lu W J, Chen J Y.Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions-Planta, 2011,234(2):377-390
[27] Wang P, Hussey P J-Interactions between plant endomembrane systems and the actin cytoskeleton-Frontiers in Plant Science,2015,6(5):422-432
[28] Porter K, Day B-From filaments to function:The role of the plant actin cytoskeleton in pathogen perception,signaling and immunity.Journal of Integrative Plant Biology, 2016, 58(4):299-311
[29]安建平,宋来庆,赵玲玲,由春香,王小非,郝玉金·苹果细胞分裂素响应因子基因MdCRF4的分离与功能鉴定·园艺学报,2017, 44(11):2055-2063An J P, Song L Q, Zhao L L, You C X, Wang X F, Hao Y J-Molecular cloning and functional characterization of a cytokinin response factor gene MdCRF4 in apple-Acta Horticulturae Sinica, 2017,44(11):2055-2063
[30]阚家亮,马娜,王彤,刘廷利,王金彦,杨郁文,蔺经,常有宏,豆梨NAC转录因子基因PcNAC1的克隆、亚细胞定位及功能初探·园艺学报,2017, 44(7):1251-1262Kan J L, Ma N, Wang T, Liu T L, Wang J Y, Yang Y W, Lin J,Chang Y H-Cloning, sub-cellular localization and functional analysis of the PcNAC 1 gene with NAC transcription factor from pyrus calleryana,Acta Horticulturae Sinica,2017,44(7):1251-1262
[31]潘琪,刘旭旭,彭浩然,蒲运丹,张永至,叶思涵,吴根土,青玲,孙现超·番茄SYTA的克隆及表达分析·中国农业科学,2017,50(15):2936-2945Pan Q, Liu X X, Peng H R, Pu Y D, Zhang Y Z, Ye S H, Wu G T, Qing L, Sun X C-Cloning, Expression analysis of Solanum lycopersicum SYTA-Scientia Agricultura Sinica,2017,50(15):2936-2945
[2]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·园艺学报,2014, 41(8):1731-1738Zhou X H, Liu J, Zhuang Y.Selection of appropriate reference genes in Solanum aculeatissimum for quantitative gene expression studies under different experimental conditions-Acta Horticulturae Sinica, 2014,41(8):1731-1738
[3]蒋晓梅,张新全,严海东,张瑜,杨盛婷,黄琳凯·柳枝稷根组织实时定量PCR分析中内参基因的选择·农业生物技术学报,2014, 22(1):55-63Jiao X M, Zhang X Q, Yan H D, Zhang Y, Yang S T, Huang L K-Reference gene selection for real-time quantitative PCR normalization in switchgrass(Panicum virgatum L.)root tissue.Journal of Agricultural Biotechnology, 2014, 22(1):55-63
[4]Wan H J,Zhao Z G,Qian C T,Chen J F.Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber.Analytical Biochemistry, 2009, 399:257-261
[5]Gutierrez L,Mauriat M,Pelloux J,Bellini C,Wuytswinkel O.Towards a systematic validation of references in real-time RTPCR.The Plant Cell, 2008,20:1734-1735
[6]Goulao L F,Fortunato A S,Ramalho J C.Selection of reference genes for normalizing quantitative real-time PCR gene expression data with multiple variables in Coffea spp-Plant Molecular Biology Reporter, 2012,30:741-759
[7] Zhu X Y, Li X P, Chen W X, Chen J Y, Lu W J, Chen L, Fu D W.Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.PLoS One,2012,7(8):e44405
[8]杨丹,李清,王贵禧,马庆华,朱利泉.平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立.中国农业科学,2017,50(12):2399-2410Yang D, Li Q, Wang Q X, Ma Q H, Zhu L Q.Reference genes selection and system establishment for real-time qPCR analysis in Ping'ou hybrid hazelnut(C.heterophylla Fisch. x C.avellana L.)-Scientia Agricultura Sinica, 2017,50(12):2399-2410
[9]Karuppaiya P,Yan X X,Liao W,Wu J,Chen F,TangL-Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas-A biodiesel plant.PLoS One,2017, 12(2):e0172460
[10] Zhang L C, Liu L, Cheng P, Shen H G, Rong B, Liu W, Yu G H.Identification and validation of reference genes for RT-qPCR analysis in banana(Musa spp.)under Fusarium wilt resistance induction conditions. Journal of Phytopathology,2017,165(11-12):746-754
[11]Fu Y,Duan X,Tang C,Li X,Voegele R T,Wang X,Wei G,Kang Z.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici.The Plant Journal, 2014, 78(1):16-30
[12] Dominguez R, Holmes K C. Actin structure and function.Annual review of biophysics, 2011,40(1):69
[13]崔力文,郑婷,张克坤,张川,上官凌飞,房经贵·葡萄Actin基因家族的鉴定及进化和表达分析·植物资源与环境学报,2017,26(3):1-10Cui L W, Zheng T, Zhang K K, Zhang C, Shangguan L F,Fang J G-Identification, evolution and expression analyses of Actin gene family of Vitis vinifera.Journal of Plant Resources and Environment, 2017,26(3):1-10
[14]李文萍,林俊城,黄科·全球花椰菜生产与贸易现状分析·中国蔬菜,2014(9):5-10Li W P,Lin J C,Huang K.Analysis about present status of global cauliflower production and its trade-China Vegetables,2014,(9):5-10
[15]顾宏辉,金昌林,赵振卿,盛小光,虞慧芳,王建升·我国松花菜产业现状及前景分析·中国蔬菜,2012(23):1-5Gu H H,Jin C L,Zhao Z Q,Sheng X G, Yu H F, Wang J S.Status quo and prospect in food industry in China-China Vegetables,2012(23):1-5
[16]丁云花,何洪巨,赵学志,王文琪,宋曙辉·不同类型花椰菜主要营养品质分析·中国蔬菜,2016(4):58-63Ding Y H, He H J, Zhao X Z, Wang W Q, Song S H.Analysis of major nutritional quality of different types of cauliflower varieties-China Vegetables, 2016(4):58-63
[17] Zhu H S, Liu J T, Wen Q F, Chen M D, Wang B, Zhang Q R,Xue ZZ, De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica'Fusi-3'fruits.PLoS One, 2017, 12(11):e0187117
[18]刘建汀,朱海生,温庆放,王彬,张前荣,陈敏氡,林珲,薛珠政.丝瓜LcWRKY21转录因子基因的克隆与表达分析·中国细胞生物学学报,2017, 39(10):1268-1278Liu J T, Zhu H S, Wen Q F, Wang B, Zhang Q R, Chen M D,Lin H, Xue Z Z.Cloning and expression analysis of transcription factor LcWRKY21 gene from Luffa cylindrical-Chinese Journal of Cell Biology, 2017, 39(10):1268-1278
[19]温庆放,刘建汀,朱海生,陈敏氡,王彬,张前荣·丝瓜过氧化氢酶基因(CAT1)的克隆及表达分析·园艺学报,2016,43(10):2039-2048Wen Q F, Liu J T, Zhu H S, Chen M D, Wang B, Zhang Q R Cloning and expression analysis of catalase CAT1 gene from Luffa cylindrical-Acta Horticulturae Sinica, 2016,43(10):2039-2048
[20]Mascia T, Santovito E,Gallitelli D,Cillo F.Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants-MolecularPlant Pathology, 2010, 11(6):805-816
[21]秦剑英,刘灶长,孔德艳,周立国,罗利军·适合于miRNA的荧光定量PCR优化体系的建立及其标准·植物遗传资源学报,2012, 13(3):435-442Qin J Y, Liu Z Z, Kong D Y, Zhou L G, Luo L J-Establishment and Criteria of Optimal Reaction System of RT-qPCR Suitable for miRNA.Journal of Plant Genetic Resources, 2012,13(3):435-442
[22]崔艳伟,李文龙,常文锁,李喜焕,张彩英·大豆异黄酮合成途径相关基因差异表达分析·植物遗传资源学报,2016, 17(4):719-725Cui Y W, Li W L, Chang W S, Li X H, Zhang C X-Differential Expression Analysis of Genes Associated with Isoflavone Synthesis in Soybean(Glycine max Merr.).Journal of Plant Genetic Resources, 2016, 17(4):719-725-
[23]刘建汀,朱海生,王彬,李永平,陈敏氡,张前荣,温庆放·西葫芦CpActin内参基因的分离及其作为内参基因的初步应用·植物遗传资源学报,2019, 20(1):188-198Liu J T,Zhu H S,Wang B,Li Y P,Chen M D,Zhang Q L,Wen Q F-Isolation of CpActin Gene and Study on This Gene as Reference Gene in cucurbita pepo. Journal of Plant Genetic Resources, 2019,20(1):188-198
[24] Udvardi M K, Czechowski T, Scheible W R-Eleven golden rules of quantitative RT-PCR-Plant Cell, 2008,20(7):1736-1737
[25] Wrzesi n ska B, Kierzek R, Obrepalska. Steplowska A.Evaluation of six commonly used reference genes for gene expression studies in herbicide-resistant Avena fatua biotypesWeed Research, 2016, 56(4):284-292
[26] Chen L, Zhong H Y, Kuang J F, Li J G, Lu W J, Chen J Y.Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions-Planta, 2011,234(2):377-390
[27] Wang P, Hussey P J-Interactions between plant endomembrane systems and the actin cytoskeleton-Frontiers in Plant Science,2015,6(5):422-432
[28] Porter K, Day B-From filaments to function:The role of the plant actin cytoskeleton in pathogen perception,signaling and immunity.Journal of Integrative Plant Biology, 2016, 58(4):299-311
[29]安建平,宋来庆,赵玲玲,由春香,王小非,郝玉金·苹果细胞分裂素响应因子基因MdCRF4的分离与功能鉴定·园艺学报,2017, 44(11):2055-2063An J P, Song L Q, Zhao L L, You C X, Wang X F, Hao Y J-Molecular cloning and functional characterization of a cytokinin response factor gene MdCRF4 in apple-Acta Horticulturae Sinica, 2017,44(11):2055-2063
[30]阚家亮,马娜,王彤,刘廷利,王金彦,杨郁文,蔺经,常有宏,豆梨NAC转录因子基因PcNAC1的克隆、亚细胞定位及功能初探·园艺学报,2017, 44(7):1251-1262Kan J L, Ma N, Wang T, Liu T L, Wang J Y, Yang Y W, Lin J,Chang Y H-Cloning, sub-cellular localization and functional analysis of the PcNAC 1 gene with NAC transcription factor from pyrus calleryana,Acta Horticulturae Sinica,2017,44(7):1251-1262
[31]潘琪,刘旭旭,彭浩然,蒲运丹,张永至,叶思涵,吴根土,青玲,孙现超·番茄SYTA的克隆及表达分析·中国农业科学,2017,50(15):2936-2945Pan Q, Liu X X, Peng H R, Pu Y D, Zhang Y Z, Ye S H, Wu G T, Qing L, Sun X C-Cloning, Expression analysis of Solanum lycopersicum SYTA-Scientia Agricultura Sinica,2017,50(15):2936-2945