芹菜β-胡萝卜素羟化酶基因AgBCH1的克隆及表达特性分析
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摘要
胡萝卜素羟化酶(β-carotene hydroxylase, BCH)在植物类胡萝卜素生物合成中起着重要的作用。芹菜是伞形科一种重要的叶菜类蔬菜作物,含有丰富的类胡萝卜素。本文以芹菜‘六合黄心芹’为实验材料,从中克隆获得编码芹菜β-胡萝卜素羟化酶的基因AgBCH1。序列分析结果显示,该基因全长933 bp,编码310个氨基酸。进化树分析表明,芹菜AgBCH1蛋白的进化高度保守,与同科的胡萝卜DcBCH1蛋白的进化关系最为相近。序列比对分析显示,植物BCH1氨基酸序列具有较高的同源性,芹菜与同科的胡萝卜BCH1氨基酸序列一致性达到87.94%。Ag BCH1蛋白相对分子质量34 505.73,理论等电点9.10,为亲水性蛋白。芹菜Ag BCH1蛋白三级蛋白结构包括5个α螺旋及6个β折叠,无序化比例为14.84%。利用实时定量PCR对经过低温(4°C)、高温(38°C)、盐(0.2mol·L-1 Na Cl)、干旱(200 g·L-1 PEG) 4种非生物胁迫处理的60 d芹菜叶片中Ag BCH1基因的相对表达量进行检测,发现Ag BCH1基因对这4种逆境胁迫均有响应,尤其对高温胁迫响应明显,且高温处理8 h后表达量最高。
carotene hydroxylase(BCH) plays an important role in the biosynthesis of plant carotenoids. Cel-ery is an important leaf vegetable crop with rich carotenoids. The AgBCH1 gene encoding β-carotene hydroxy-lase was cloned from celery ‘Liuhe Huangxinqin'. Sequence analysis showed that the open reading frame ofAgBCH1 gene was 933 bp, which encoded 310 amino acids. Phylogenetic analysis indicates that the BCHshowed high evolutionary conservation, and the AgBCH1 was most closed to Daucus carota. The homology of BCHwas up to 87.94% between celery and carrot. The relative molecular mass of AgBCH1 protein was 34 505.73, andthe theoretical isoelectric point was 9.10. The physicochemical property analysis showed that AgBCH belongedto hydrophilic protein. There were 5 alpha helices and 6 beta folds in the predicted tertiary structure of the pro-tein. The results of disordered analysis showed that the disordering ratio was 14.84%. The relative expressionlevels of AgBCH1 gene in celery under abiotic stress treatments(low temperature, high temperature, salt stress,and drought) were detected by real-time quantitative PCR. The results showed that AgBCH1 gene was responseto abiotic stress, which was obvious under high temperature stress. The expression level of AgBCH1 gene wasthe highest after 8 h under high temperature stress.
carotene hydroxylase(BCH) plays an important role in the biosynthesis of plant carotenoids. Cel-ery is an important leaf vegetable crop with rich carotenoids. The AgBCH1 gene encoding β-carotene hydroxy-lase was cloned from celery ‘Liuhe Huangxinqin'. Sequence analysis showed that the open reading frame ofAgBCH1 gene was 933 bp, which encoded 310 amino acids. Phylogenetic analysis indicates that the BCHshowed high evolutionary conservation, and the AgBCH1 was most closed to Daucus carota. The homology of BCHwas up to 87.94% between celery and carrot. The relative molecular mass of AgBCH1 protein was 34 505.73, andthe theoretical isoelectric point was 9.10. The physicochemical property analysis showed that AgBCH belongedto hydrophilic protein. There were 5 alpha helices and 6 beta folds in the predicted tertiary structure of the pro-tein. The results of disordered analysis showed that the disordering ratio was 14.84%. The relative expressionlevels of AgBCH1 gene in celery under abiotic stress treatments(low temperature, high temperature, salt stress,and drought) were detected by real-time quantitative PCR. The results showed that AgBCH1 gene was responseto abiotic stress, which was obvious under high temperature stress. The expression level of AgBCH1 gene wasthe highest after 8 h under high temperature stress.
引文
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Bouvier F,Keller Y,D'Harlingue A,et al(1998).Xanthophyll biosynthesis:molecular and functional characterization of carotenoid hydroxylases from pepper fruits(Capsicum annuum L.).Biochim Biophys Acta,1391:320-328
Boyer JS(1982).Plant productivity and environment.Science,218(4571):443-448
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Dianat M,Veisi A,Ahangarpour A,et al(2015).The effect of hydro-alcoholic celery(Apium graveolens)leaf extract on cardiovascular parameters and lipid profile in animal model of hypertension induced by fructose.Avicenna JPhytomed,5(3):203-209
Diretto G,Welsch R,Tavazza R,et al(2007).Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.BMC Plant Biol,7:11
Du H,Wang N,Cui F,et al(2010).Characterization of aβ-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice.Plant Physiol,154(3):1304-1318
Feng K,Hou XL,Li MY,et al(2018).CeleryDB:a genomic database for celery.Database(Oxford),doi:10.1093/database/bay070
Feng K,Wang GL,Huang Y,et al(2016).Cloning and expression analysis of gibberellin oxidase gene AgGA2ox from Apium graceolens under abiotic stress treatments.Plant Physiol J,52(6):948-956(in Chinese with English abstract)[冯凯,王广龙,黄莹等(2016).芹菜赤霉素氧化酶基因AgGA2ox的克隆及非生物逆境下表达分析.植物生理学报,52(6):948-956]
Feng TK,Li SG,Wang YL,et al(2007).Cloning and sequence analysis ofβ-carotene hydroxylase gene from Nanfeng orange(Citrus reticulata Blanco var kinokuni(Tanaka)H H Hu).Acta Bot Boreal-Occident Sin,27(2):238-243(in Chinese with English abstract)[冯唐锴,李思光,汪艳璐等(2007).南丰蜜橘β-胡萝卜素羟化酶基因的克隆和序列分析.西北植物学报,27(2):238-243]
Gasteiger E,Gattiker A,Hoogland C,et al(2003).ExPASy:the proteomics server for in-depth protein knowledge and analysis.Nucleic Acids Res,31(13):3784-3788
Kim IJ,Ko KC,Kim CS,et al(2001).Isolation and characterization of cDNAs encodingβ-carotene hydroxylase in Citrus.Plant Sci,161(5):1005-1010
Kim SH,Ahn YO,Ahn MJ,et al(2012).Down-regulation ofβ-carotene hydroxylase increasesβ-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato.Phytochemistry,74:69-78
Lagarde D,Vermaas W(1999).The zeaxanthin biosynthesis enzymeβ-carotene hydroxylase is involved in myxoxanthophyll synthesis in Synechocystis sp.PCC 6803.FEBSLett,454(3):247-251
Li JW,Ma J,Feng K,et al(2018a).Carotenoid accumulation and distinct transcript profiling of structural genes involved in carotenoid biosynthesis in celery.Plant Mol Biol Rep,36(4):663-674
Li MY,Hou XL,Wang F,et al(2018b).Advances in the research of celery,an important Apiaceae vegetable crop.Crit Rev Biotechnol,38(2):172-183
Li MY,Wang F,Jiang Q,et al(2014).Identification of SSRs and differentially expressed genes in two cultivars of celery(Apium graveolens L.)by deep transcriptome sequencing.Hortic Res,1:10
Li MY,Wang F,Jiang Q,et al(2016).Validation and comparison of reference genes for qPCR normalization of celery(Apium graveolens)at different development stages.Front Plant Sci,7:313
Li Q,Farre G,Naqvi S,et al(2010).Cloning and functional characterization of the maize carotenoid isomerase andβ-carotene hydroxylase genes and their regulation during endosperm maturation.Transgenic Res,19(6):1053-1068
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25:402-408
Mochimaru M,Masukawa H,Maoka T,et al(2008).Substrate specificities and availability of fucosyltransferase andβ-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp.strain PCC 7120 compared with Synechocystis sp.strain PCC 6803.J Bacteriol,190(20):6726-6733
Muthusamy V,Hossain F,Thirunavukkarasu N,et al(2014).Development ofβ-carotene rich maize hybrids through marker-assisted introgression ofβ-carotene hydroxylase allele.PLoS One,9(12):e113583
Nisar N,Li L,Lu S,et al(2015).Carotenoid metabolism in plants.Mol Plant,8(1):68-82
Pogson BJ,Rissler HM(2000).Genetic manipulation of carotenoid biosynthesis and photoprotection.Philos Trans R Soc Lond B Biol Sci,355(1402):1395-1403
Sun HY,Chen Y,Zhao HS,et al(2015).Molecular characteristics and functional analysis ofβ-carotene hydroxylase gene from phyllostachys edulis.Sci Silv Sin,51(10):53-59(in Chinese with English abstract)[孙化雨,陈颖,赵韩生等(2015).毛竹β-胡萝卜素羟化酶基因的分子特征及其功能.林业科学,51(10):53-59]
Sun Z,Gantt E,Cunningham FX(1996).Cloning and functional analysis of theβ-carotene hydroxylase of Arabidopsis thaliana.J Biol Chem,271(40):24349-24352
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28(10):2731-2739
Tan CP,Zhao FQ,Su ZL,et al(2007).Expression ofβ-carotene hydroxylase gene(crtR-B)from the green alga Haematococcus pluvialis,in chloroplasts of Chlamydomonas reinhardtii.J Appl Phycol,19(4):347-355
Tian L,Magallanes-Lundback M,Musetti V,et al(2003).Functional analysis ofβ-and?-ring carotenoid hydroxylases in Arabidopsis.Plant Cell,15(6):1320-1332
Valliyodan B,Nguyen HT(2006).Understanding regulatory networks and engineering for enhanced drought tolerance in plants.Curr Opin Plant Biol,9(2):189-195
Verslues PE,Bray EA(2006).Role of abscisic acid(ABA)and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation.J Exp Bot,57(1):201-212
Xu F,Gong YF,Liu L,et al(2015).Acquisition and salt-tolerance evaluation of transgenic tobacco(Nicotiana tabacum)withβ-carotene hydroxylase gene(chyb)from Dunaliella viridis.J Agric Biotech,23(9):1149-1156(in Chinese with English abstract)[许峰,龚一富,刘林等(2015).转盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)烟草的获得及耐盐性鉴定.农业生物技术学报,23(9):1149-1156]
Yamagishi M,Kishimoto S,Nakayama M(2010).Carotenoid composition and changes in expression of carotenoid biosynthetic genes in tepals of Asiatic hybrid lily.Plant Breeding,129(1):100-107
Zhang JC,Zhou WJ,Xu Q,et al(2013).Two lycopeneβ-cyclases genes from sweet orange(Citrus sinensis L.Osbeck)encode enzymes with different functional efficiency during the conversion of lycopene-to-provitamin A.JIntegr Agr,12(10):1731-1747
Zhu JK(2016).Abiotic stress signaling and responses in plants.Cell,167(2):313-324
Bouvier F,Keller Y,D'Harlingue A,et al(1998).Xanthophyll biosynthesis:molecular and functional characterization of carotenoid hydroxylases from pepper fruits(Capsicum annuum L.).Biochim Biophys Acta,1391:320-328
Boyer JS(1982).Plant productivity and environment.Science,218(4571):443-448
Davison PA,Hunter CN,Horton P(2002).Overexpression of beta-carotene hydroxylase enhances stress tolerance in Arabidopsis.Nature,418(6894):203-206
Demmig-Adams B,Gilmore AM,Adams WW(1996).Carotenoids 3:in vivo function of carotenoids in higher plants.FASEB J,10(4):403-412
Dianat M,Veisi A,Ahangarpour A,et al(2015).The effect of hydro-alcoholic celery(Apium graveolens)leaf extract on cardiovascular parameters and lipid profile in animal model of hypertension induced by fructose.Avicenna JPhytomed,5(3):203-209
Diretto G,Welsch R,Tavazza R,et al(2007).Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.BMC Plant Biol,7:11
Du H,Wang N,Cui F,et al(2010).Characterization of aβ-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice.Plant Physiol,154(3):1304-1318
Feng K,Hou XL,Li MY,et al(2018).CeleryDB:a genomic database for celery.Database(Oxford),doi:10.1093/database/bay070
Feng K,Wang GL,Huang Y,et al(2016).Cloning and expression analysis of gibberellin oxidase gene AgGA2ox from Apium graceolens under abiotic stress treatments.Plant Physiol J,52(6):948-956(in Chinese with English abstract)[冯凯,王广龙,黄莹等(2016).芹菜赤霉素氧化酶基因AgGA2ox的克隆及非生物逆境下表达分析.植物生理学报,52(6):948-956]
Feng TK,Li SG,Wang YL,et al(2007).Cloning and sequence analysis ofβ-carotene hydroxylase gene from Nanfeng orange(Citrus reticulata Blanco var kinokuni(Tanaka)H H Hu).Acta Bot Boreal-Occident Sin,27(2):238-243(in Chinese with English abstract)[冯唐锴,李思光,汪艳璐等(2007).南丰蜜橘β-胡萝卜素羟化酶基因的克隆和序列分析.西北植物学报,27(2):238-243]
Gasteiger E,Gattiker A,Hoogland C,et al(2003).ExPASy:the proteomics server for in-depth protein knowledge and analysis.Nucleic Acids Res,31(13):3784-3788
Kim IJ,Ko KC,Kim CS,et al(2001).Isolation and characterization of cDNAs encodingβ-carotene hydroxylase in Citrus.Plant Sci,161(5):1005-1010
Kim SH,Ahn YO,Ahn MJ,et al(2012).Down-regulation ofβ-carotene hydroxylase increasesβ-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato.Phytochemistry,74:69-78
Lagarde D,Vermaas W(1999).The zeaxanthin biosynthesis enzymeβ-carotene hydroxylase is involved in myxoxanthophyll synthesis in Synechocystis sp.PCC 6803.FEBSLett,454(3):247-251
Li JW,Ma J,Feng K,et al(2018a).Carotenoid accumulation and distinct transcript profiling of structural genes involved in carotenoid biosynthesis in celery.Plant Mol Biol Rep,36(4):663-674
Li MY,Hou XL,Wang F,et al(2018b).Advances in the research of celery,an important Apiaceae vegetable crop.Crit Rev Biotechnol,38(2):172-183
Li MY,Wang F,Jiang Q,et al(2014).Identification of SSRs and differentially expressed genes in two cultivars of celery(Apium graveolens L.)by deep transcriptome sequencing.Hortic Res,1:10
Li MY,Wang F,Jiang Q,et al(2016).Validation and comparison of reference genes for qPCR normalization of celery(Apium graveolens)at different development stages.Front Plant Sci,7:313
Li Q,Farre G,Naqvi S,et al(2010).Cloning and functional characterization of the maize carotenoid isomerase andβ-carotene hydroxylase genes and their regulation during endosperm maturation.Transgenic Res,19(6):1053-1068
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25:402-408
Mochimaru M,Masukawa H,Maoka T,et al(2008).Substrate specificities and availability of fucosyltransferase andβ-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp.strain PCC 7120 compared with Synechocystis sp.strain PCC 6803.J Bacteriol,190(20):6726-6733
Muthusamy V,Hossain F,Thirunavukkarasu N,et al(2014).Development ofβ-carotene rich maize hybrids through marker-assisted introgression ofβ-carotene hydroxylase allele.PLoS One,9(12):e113583
Nisar N,Li L,Lu S,et al(2015).Carotenoid metabolism in plants.Mol Plant,8(1):68-82
Pogson BJ,Rissler HM(2000).Genetic manipulation of carotenoid biosynthesis and photoprotection.Philos Trans R Soc Lond B Biol Sci,355(1402):1395-1403
Sun HY,Chen Y,Zhao HS,et al(2015).Molecular characteristics and functional analysis ofβ-carotene hydroxylase gene from phyllostachys edulis.Sci Silv Sin,51(10):53-59(in Chinese with English abstract)[孙化雨,陈颖,赵韩生等(2015).毛竹β-胡萝卜素羟化酶基因的分子特征及其功能.林业科学,51(10):53-59]
Sun Z,Gantt E,Cunningham FX(1996).Cloning and functional analysis of theβ-carotene hydroxylase of Arabidopsis thaliana.J Biol Chem,271(40):24349-24352
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28(10):2731-2739
Tan CP,Zhao FQ,Su ZL,et al(2007).Expression ofβ-carotene hydroxylase gene(crtR-B)from the green alga Haematococcus pluvialis,in chloroplasts of Chlamydomonas reinhardtii.J Appl Phycol,19(4):347-355
Tian L,Magallanes-Lundback M,Musetti V,et al(2003).Functional analysis ofβ-and?-ring carotenoid hydroxylases in Arabidopsis.Plant Cell,15(6):1320-1332
Valliyodan B,Nguyen HT(2006).Understanding regulatory networks and engineering for enhanced drought tolerance in plants.Curr Opin Plant Biol,9(2):189-195
Verslues PE,Bray EA(2006).Role of abscisic acid(ABA)and Arabidopsis thaliana ABA-insensitive loci in low water potential-induced ABA and proline accumulation.J Exp Bot,57(1):201-212
Xu F,Gong YF,Liu L,et al(2015).Acquisition and salt-tolerance evaluation of transgenic tobacco(Nicotiana tabacum)withβ-carotene hydroxylase gene(chyb)from Dunaliella viridis.J Agric Biotech,23(9):1149-1156(in Chinese with English abstract)[许峰,龚一富,刘林等(2015).转盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)烟草的获得及耐盐性鉴定.农业生物技术学报,23(9):1149-1156]
Yamagishi M,Kishimoto S,Nakayama M(2010).Carotenoid composition and changes in expression of carotenoid biosynthetic genes in tepals of Asiatic hybrid lily.Plant Breeding,129(1):100-107
Zhang JC,Zhou WJ,Xu Q,et al(2013).Two lycopeneβ-cyclases genes from sweet orange(Citrus sinensis L.Osbeck)encode enzymes with different functional efficiency during the conversion of lycopene-to-provitamin A.JIntegr Agr,12(10):1731-1747
Zhu JK(2016).Abiotic stress signaling and responses in plants.Cell,167(2):313-324