苹果根皮苷-2-O-糖基转移酶基因克隆与表达模式分析
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摘要
目的:根皮苷-2-O-糖基转移酶(phloridzin 2’-O-glycosyltransferase,P2’-GT)是根皮苷合成最后一步关键酶,可以把根皮素转化成根皮苷,本研究在富士苹果中克隆出P2’-GT基因,对基因编码产物进行生物信息学分析和基因的表达模式分析。方法:以苹果皮为材料,提取RNA反转录合成cDNA为模板,设计特异性引物进行扩增和测序,利用Pro Param tool、TMHMM等在线软件对编码蛋白进行生物信息学分析,利用实时荧光定量聚合酶链式反应(realtime?uorescence quatitative polymerase chain reaction,RTFQ-PCR)分析P2’-GT在苹果不同部位、不同苹果品种和不同生长时期的表达差异。结果:P2’-GT cDNA全长1 452 bp,编码483个氨基酸,相对分子质量53.6 kDa,等电点5.76,该基因编码蛋白是不稳定蛋白,不具有明显的跨膜结构,二级结构主要有α-螺旋、无规卷曲和延伸链组成,三级结构结果显示P2’-GT蛋白模型与对苯二酚葡萄糖基转移酶相似度最高(41.32%),进化分析表明P2’-GT与白梨糖基转移酶同源性最高。RTFQ-PCR分析发现P2’-GT在3种苹果皮中均高效表达,在叶和根中微量表达,在果肉中不表达;P2’-GT的转录表达受苹果发育调控,在生长初期几乎不表达,随着苹果发育表达量逐渐增加,在生长中期达到最高值,随后开始减少,至苹果成熟期下降到最高值的50%水平。此外,P2’-GT在3种苹果品种中表达不同,在澳洲青苹中表达量最高,在嘎啦中表达量最低。结论:本研究明确了P2’-GT的生物信息学特性及P2’-GT基因在不同生长期和不同部位的表达差异,为调控根皮苷合成的研究提供理论依据。
Objective: Phloridzin 2-O-glucosyltransferase(P2'-GT) is the key enzyme involved in the final step in the phloridzin biosynthetic pathway, catalyzing transformation of phloretin into phlorizin. The purpose of this study was to clone a P2'-GT gene from Fuji apple and to investigate bioinformatics analysis of the protein encoded by the gene and its expression pattern. Methods: RNA was extracted from apple peel and was used to synthesize a cDNA template by reverse transcription. Speci?c primers were designed based on the P2'-GT reporter gene for PCR ampli?cation and sequencing.Bioinformatic analyses were performed using online software(Pro Param tool and TMHMM). The differential transcript levels of P2'-GT in different organs of apple varieties at different growth stages were detected using real-time ?uorescence quantitative polymerase chain reaction(RTFQ-PCR). Results: The cDNA was 1 452 bp long, encoding 483 amino acid residues. The encoded protein was unstable with a molecular mass of 53.6 kDa and a theoretical PI of 5.76. In addition, the protein had no obvious transmembrane structure. Its secondary structure consisted of α-helix, random coil and extended strand. The protein tertiary structure exhibited high similarity to hydroquinone glucosyltransferase(41.32%). Phylogenetic analysis indicated that P2'-GT had the highest homology to Pyrus bretschneideri glucosyltransferase. The expression level of this gene was high in apple peel but extremely low in leaves and roots and zero in apple pulp. P2'-GT gene expression was related to the growth period; transcript level was rarely detectable in the early stage, but increased rapidly to a maximum in the middle stage and then decreased by 50% in the maturation stage. Signi?cant differences in the transcript level of this gene were found among three different varieties, with the highest and lowest level being observed in Granny Smith and Gala,respectively. This research provides theoretical support for the role of P2'-GT genes in regulating the synthesis of phloridzin.
Objective: Phloridzin 2-O-glucosyltransferase(P2'-GT) is the key enzyme involved in the final step in the phloridzin biosynthetic pathway, catalyzing transformation of phloretin into phlorizin. The purpose of this study was to clone a P2'-GT gene from Fuji apple and to investigate bioinformatics analysis of the protein encoded by the gene and its expression pattern. Methods: RNA was extracted from apple peel and was used to synthesize a cDNA template by reverse transcription. Speci?c primers were designed based on the P2'-GT reporter gene for PCR ampli?cation and sequencing.Bioinformatic analyses were performed using online software(Pro Param tool and TMHMM). The differential transcript levels of P2'-GT in different organs of apple varieties at different growth stages were detected using real-time ?uorescence quantitative polymerase chain reaction(RTFQ-PCR). Results: The cDNA was 1 452 bp long, encoding 483 amino acid residues. The encoded protein was unstable with a molecular mass of 53.6 kDa and a theoretical PI of 5.76. In addition, the protein had no obvious transmembrane structure. Its secondary structure consisted of α-helix, random coil and extended strand. The protein tertiary structure exhibited high similarity to hydroquinone glucosyltransferase(41.32%). Phylogenetic analysis indicated that P2'-GT had the highest homology to Pyrus bretschneideri glucosyltransferase. The expression level of this gene was high in apple peel but extremely low in leaves and roots and zero in apple pulp. P2'-GT gene expression was related to the growth period; transcript level was rarely detectable in the early stage, but increased rapidly to a maximum in the middle stage and then decreased by 50% in the maturation stage. Signi?cant differences in the transcript level of this gene were found among three different varieties, with the highest and lowest level being observed in Granny Smith and Gala,respectively. This research provides theoretical support for the role of P2'-GT genes in regulating the synthesis of phloridzin.
引文
[1]TANG J,TANG L,TAN S.The study of variation of phloridzin content in six wild malus species[J].Journal of Food and Nutrition Research,2015,3(3):146-151.DOI:10.12691/jfnr-3-3-3.
[2]XU L J,GUO J R,CHEN Q Q,et al.Quantitation of phlorizin and phloretin using an ultra high performance liquid chromatographyelectrospray ionization tandem mass spectrometric method[J].Journal of Chromatography B,2014,960(1):67-72.DOI:10.1016/j.jchromb.2014.04.007.
[3]FROMM M,BAYHA S,CARLE R,et al.Characterization and quantitation of low and high molecular weight phenolic compounds in apple seeds[J].Journal of Agricultural and Food Chemistry,2012,60(5):1232-1242.DOI:10.1021/jf204623d.
[4]VEITCH N C,GRAYER R J.Chemlnform abstract:flavonoids and their glycosides,including anthocyanins[J].Natural Product Reports,2008,39(39):1626-1695.DOI:10.1002/chin.200839260.
[5]DE BERNONVILLE T D,GAUCHER M,GUYOT S,et al.The constitutive phenolic composition of two Malus×domestica genotypes is not responsible for their contrasted susceptibilities to fire blight[J].Environmental and Experimental Botany,2011,74:65-73.DOI:10.1016/j.envexpbot.2011.04.019.
[6]WANG Q,QIU L,CHEN X R,et al.Inhibitory effects of phloridzin dihydrate on the activity of mushroom(Agaricus bisporus)tyrosinase[J].Bioorganic&Medicinal Chemistry,2007,15(3):1568-1571.DOI:10.1016/j.bmc.2005.11.048.
[7]EHRENKRANZ J R L,LEWIS N G,KAHN C R,et al.Phlorizin:a review[J].Diabetes/Metablism Research Reviews,2005,21(1):31-38.
[8]PEI F,LI B Y,ZHANG Z,et al.Beneficial effect of phlorizin on diabetic nephropathy in diabetic db/db mice[J].Journal of Diabetes and Its Complications,2014,28(5):596-603.DOI:10.1016/j.jdiacomp.2014.04.010.
[9]ZHANG X Z,ZHAO Y B,LI C M,et al.Potential polyphenol markers of phase change in apple(Malus domestica)[J].Journal of Plant Physiology,2007,164(5):574-580.DOI:10.1016/j.jplph.2006.03.011.
[10]HOFMANN A,WITTENMAYER L,ARNOLD G,et al.Root exudation of phloridzin by apple seedlings(Malus×domestica Borkh.)with symptoms of apple replant disease[J].Journal of Applied Botany and Food Quality,2009,82(2):193-198.
[11]LEMOINE R,DELROT S.Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves[J].Physiologia Plantarum,1987,69(4):639-644.DOI:10.1111/j.1399-3054.1987.tb01978.x.
[12]DIMITROVSKA M,BOCEVSKA M,DIMITROVSKI D,et al.Anthocyanin composition of Vranec,Cabernet Sauvignon,Merlot and Pinot Noir grapes as indicator of their varietal differentiation[J].European Food Research and Technology,2011,232(4):591-600.DOI:10.1007/s00217-011-1425-9.
[13]FANZONE M,ZAMORA F,JOFRéV,et al.Phenolic composition of malbec grape skins and seeds from valle de uco(Mendoza,Argentina)during ripening effect of cluster thinning[J].Journal of Agricultural and Food Chemistry,2011,59(11):6120-6136.DOI:10.1021/jf200073k.
[14]BERLI F J,FANZONE M,PICCOLI P,at al.Solar UV-B and ABAare involved in phenol metabolism of Vitis vinifera L.increasing biosynthesis of berry skin polyphenols[J].Journal of Agricultural and Food Chemistry,2011,59(9):4874-4884.DOI:10.1021/jf200040z.
[15]GONZáLEZ M F,CARBALLO E M,GRANDE B C,et al.Pattern recognition of three Vitis vinifera L.red grapes varieties based on anthocyanin and flavonol profiles,with correlations between their biosynthesis pathways[J].Food Chemistry,2012,130(1):9-19.DOI:10.1016/j.foodchem.2011.06.006.
[16]ANDERSEN M O,MARKHAM K R.Flavonoids:chemistry,biochemistry and applications[M].Taylor&Francis Group,USA,2006.
[17]ALMEIDA J R M,D’AMICO E,PREUSS A,et al.Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry(Fragaria×ananassa)[J].Archives of Biochemistry&Biophysics,2007,465(1):61-71.DOI:10.1016/j.abb.2007.04.040.
[18]B?NISCH F,FROTSCHER J,STANITZEK S,et al.Activity-based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from grape[J].Plant Physiology,2014,166(1):23-39.DOI:10.1104/pp.114.242578.
[19]FERREYRA M L F,RODRIGUEZ E,CASAS M I,et al.Identification of a bifunctional maize C-and O-glucosyltransferase[J].Journal of Biological Chemistry,2013,288(44):31678-31688.DOI:10.1074/jbc.M113.510040.
[20]胡德龙.茄子花青素糖基转移酶基因SmGT的克隆与生物信息学分析[J].浙江农业科学,2017,58(11):2029-2033.DOI:10.16178/j.issn.0528-9017.20171149.
[21]燕一波,郭玉双.金鱼草花青素糖基转移酶基因的克隆与表达特性分析[J].热带作物学报,2017,38(6):1101-1105.DOI:10.3969/j.issn.1000-2561.2017.06.019.
[22]NOGUCHI A,SAITO A,YU H,et al.A UDP-glucose:isoflavone7-O-glucosyltransferase from the roots of soybean(Glycine max)seedlings[J].Journal of Biological Chemistry,2007,282(32):23581-23590.DOI:10.1074/jbc.M702651200.
[23]罗少华,牛涛,苏香萍,等.鲜人参皂苷β-葡萄糖苷酶生物转化研究[J].时珍国医国药,2012,23(1):106-108.
[24]D AV I E S K M,M A R S H A L L G B,B R A D L E Y J M,e t a l.Characterisation of aurone biosynthesis in Antirrhinum majus[J].Physiologia Plantarum,2006,128(4):593-603.DOI:10.1111/j.1399-3054.2006.00774.x.
[25]OGATA J,KANNO Y,ITOH Y,et al.Plant biochemistry:anthocyanin biosynthesis in roses[J].Nature,2005,435:757-758.DOI:10.1038/nature435757a.
[26]刘吉升,吴璇,吕旻,等.葛根糖基转移酶蛋白肽段序列的分离与鉴定[J].生物技术,2011,21(1):4-7.
[27]WANG X,FAN R,LI J,et al.Molecular cloning and functional characterization of a novel(iso)flavone 4′,7-O-diglucoside glucosyltransferase from Pueraria lobata[J].Frontiers in Plant Science,2016,7:387-398.DOI:10.3389/fpls.2016.00387.
[28]莫长明,马小军,唐其,等.罗汉果葡萄糖基转移酶基因SgUGT4的克隆及表达研究[J].园艺学报,2015,34(3):523-534.DOI:10.16420/j.issn.0513-353x.2014-0757.
[29]徐颖,樊明涛,李亚辉,等.苹果根皮苷糖基转移酶基因克隆及生物信息学分析[J].中国食品学报,2016,16(11):204-211.DOI:10.16429/j.1009-7848.2016.11.028.
[30]DARE A P,YAUK Y K,TOMES S,et al.Silencing a phloretinspecific glycosyltransferase perturbs both general phenylpropanoid biosynthesis and plant development[J].Plant Journal for Cell&Molecular Biology,2017,91(2):237-250.DOI:10.1111/tpj.13559.
[31]杜灵娟,陈凯利,刘雅莉.葡萄风信子FLS1基因克隆及其表达与花色性状之间的关联性分析[J].西北林学院学报,2017,32(1):106-113.DOI:10.3969/j.issn.1001-7461.2017.01.17.
[32]冉军舰,徐剑宏,赫丹,等.小麦赤霉病原菌拮抗菌B a c i l l u s amyloliquefaciens 7M1产抗菌素的研究[J].微生物学通报,2016,43(11):2437-2447.
[33]GE J,SUN Y,XIN X,et al.Purification and partial characterization of a novel bacteriocin synthesized by Lactobacillus paracasei HD1-7isolated from Chinese sauerkraut juice[J].Scientific Reports,2016,6:19366.DOI:10.1038/srep19366.
[34]冉军舰.苹果多酚的组分鉴定及功能特性研究[D].杨凌:西北农林科技大学,2013.
[2]XU L J,GUO J R,CHEN Q Q,et al.Quantitation of phlorizin and phloretin using an ultra high performance liquid chromatographyelectrospray ionization tandem mass spectrometric method[J].Journal of Chromatography B,2014,960(1):67-72.DOI:10.1016/j.jchromb.2014.04.007.
[3]FROMM M,BAYHA S,CARLE R,et al.Characterization and quantitation of low and high molecular weight phenolic compounds in apple seeds[J].Journal of Agricultural and Food Chemistry,2012,60(5):1232-1242.DOI:10.1021/jf204623d.
[4]VEITCH N C,GRAYER R J.Chemlnform abstract:flavonoids and their glycosides,including anthocyanins[J].Natural Product Reports,2008,39(39):1626-1695.DOI:10.1002/chin.200839260.
[5]DE BERNONVILLE T D,GAUCHER M,GUYOT S,et al.The constitutive phenolic composition of two Malus×domestica genotypes is not responsible for their contrasted susceptibilities to fire blight[J].Environmental and Experimental Botany,2011,74:65-73.DOI:10.1016/j.envexpbot.2011.04.019.
[6]WANG Q,QIU L,CHEN X R,et al.Inhibitory effects of phloridzin dihydrate on the activity of mushroom(Agaricus bisporus)tyrosinase[J].Bioorganic&Medicinal Chemistry,2007,15(3):1568-1571.DOI:10.1016/j.bmc.2005.11.048.
[7]EHRENKRANZ J R L,LEWIS N G,KAHN C R,et al.Phlorizin:a review[J].Diabetes/Metablism Research Reviews,2005,21(1):31-38.
[8]PEI F,LI B Y,ZHANG Z,et al.Beneficial effect of phlorizin on diabetic nephropathy in diabetic db/db mice[J].Journal of Diabetes and Its Complications,2014,28(5):596-603.DOI:10.1016/j.jdiacomp.2014.04.010.
[9]ZHANG X Z,ZHAO Y B,LI C M,et al.Potential polyphenol markers of phase change in apple(Malus domestica)[J].Journal of Plant Physiology,2007,164(5):574-580.DOI:10.1016/j.jplph.2006.03.011.
[10]HOFMANN A,WITTENMAYER L,ARNOLD G,et al.Root exudation of phloridzin by apple seedlings(Malus×domestica Borkh.)with symptoms of apple replant disease[J].Journal of Applied Botany and Food Quality,2009,82(2):193-198.
[11]LEMOINE R,DELROT S.Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves[J].Physiologia Plantarum,1987,69(4):639-644.DOI:10.1111/j.1399-3054.1987.tb01978.x.
[12]DIMITROVSKA M,BOCEVSKA M,DIMITROVSKI D,et al.Anthocyanin composition of Vranec,Cabernet Sauvignon,Merlot and Pinot Noir grapes as indicator of their varietal differentiation[J].European Food Research and Technology,2011,232(4):591-600.DOI:10.1007/s00217-011-1425-9.
[13]FANZONE M,ZAMORA F,JOFRéV,et al.Phenolic composition of malbec grape skins and seeds from valle de uco(Mendoza,Argentina)during ripening effect of cluster thinning[J].Journal of Agricultural and Food Chemistry,2011,59(11):6120-6136.DOI:10.1021/jf200073k.
[14]BERLI F J,FANZONE M,PICCOLI P,at al.Solar UV-B and ABAare involved in phenol metabolism of Vitis vinifera L.increasing biosynthesis of berry skin polyphenols[J].Journal of Agricultural and Food Chemistry,2011,59(9):4874-4884.DOI:10.1021/jf200040z.
[15]GONZáLEZ M F,CARBALLO E M,GRANDE B C,et al.Pattern recognition of three Vitis vinifera L.red grapes varieties based on anthocyanin and flavonol profiles,with correlations between their biosynthesis pathways[J].Food Chemistry,2012,130(1):9-19.DOI:10.1016/j.foodchem.2011.06.006.
[16]ANDERSEN M O,MARKHAM K R.Flavonoids:chemistry,biochemistry and applications[M].Taylor&Francis Group,USA,2006.
[17]ALMEIDA J R M,D’AMICO E,PREUSS A,et al.Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry(Fragaria×ananassa)[J].Archives of Biochemistry&Biophysics,2007,465(1):61-71.DOI:10.1016/j.abb.2007.04.040.
[18]B?NISCH F,FROTSCHER J,STANITZEK S,et al.Activity-based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from grape[J].Plant Physiology,2014,166(1):23-39.DOI:10.1104/pp.114.242578.
[19]FERREYRA M L F,RODRIGUEZ E,CASAS M I,et al.Identification of a bifunctional maize C-and O-glucosyltransferase[J].Journal of Biological Chemistry,2013,288(44):31678-31688.DOI:10.1074/jbc.M113.510040.
[20]胡德龙.茄子花青素糖基转移酶基因SmGT的克隆与生物信息学分析[J].浙江农业科学,2017,58(11):2029-2033.DOI:10.16178/j.issn.0528-9017.20171149.
[21]燕一波,郭玉双.金鱼草花青素糖基转移酶基因的克隆与表达特性分析[J].热带作物学报,2017,38(6):1101-1105.DOI:10.3969/j.issn.1000-2561.2017.06.019.
[22]NOGUCHI A,SAITO A,YU H,et al.A UDP-glucose:isoflavone7-O-glucosyltransferase from the roots of soybean(Glycine max)seedlings[J].Journal of Biological Chemistry,2007,282(32):23581-23590.DOI:10.1074/jbc.M702651200.
[23]罗少华,牛涛,苏香萍,等.鲜人参皂苷β-葡萄糖苷酶生物转化研究[J].时珍国医国药,2012,23(1):106-108.
[24]D AV I E S K M,M A R S H A L L G B,B R A D L E Y J M,e t a l.Characterisation of aurone biosynthesis in Antirrhinum majus[J].Physiologia Plantarum,2006,128(4):593-603.DOI:10.1111/j.1399-3054.2006.00774.x.
[25]OGATA J,KANNO Y,ITOH Y,et al.Plant biochemistry:anthocyanin biosynthesis in roses[J].Nature,2005,435:757-758.DOI:10.1038/nature435757a.
[26]刘吉升,吴璇,吕旻,等.葛根糖基转移酶蛋白肽段序列的分离与鉴定[J].生物技术,2011,21(1):4-7.
[27]WANG X,FAN R,LI J,et al.Molecular cloning and functional characterization of a novel(iso)flavone 4′,7-O-diglucoside glucosyltransferase from Pueraria lobata[J].Frontiers in Plant Science,2016,7:387-398.DOI:10.3389/fpls.2016.00387.
[28]莫长明,马小军,唐其,等.罗汉果葡萄糖基转移酶基因SgUGT4的克隆及表达研究[J].园艺学报,2015,34(3):523-534.DOI:10.16420/j.issn.0513-353x.2014-0757.
[29]徐颖,樊明涛,李亚辉,等.苹果根皮苷糖基转移酶基因克隆及生物信息学分析[J].中国食品学报,2016,16(11):204-211.DOI:10.16429/j.1009-7848.2016.11.028.
[30]DARE A P,YAUK Y K,TOMES S,et al.Silencing a phloretinspecific glycosyltransferase perturbs both general phenylpropanoid biosynthesis and plant development[J].Plant Journal for Cell&Molecular Biology,2017,91(2):237-250.DOI:10.1111/tpj.13559.
[31]杜灵娟,陈凯利,刘雅莉.葡萄风信子FLS1基因克隆及其表达与花色性状之间的关联性分析[J].西北林学院学报,2017,32(1):106-113.DOI:10.3969/j.issn.1001-7461.2017.01.17.
[32]冉军舰,徐剑宏,赫丹,等.小麦赤霉病原菌拮抗菌B a c i l l u s amyloliquefaciens 7M1产抗菌素的研究[J].微生物学通报,2016,43(11):2437-2447.
[33]GE J,SUN Y,XIN X,et al.Purification and partial characterization of a novel bacteriocin synthesized by Lactobacillus paracasei HD1-7isolated from Chinese sauerkraut juice[J].Scientific Reports,2016,6:19366.DOI:10.1038/srep19366.
[34]冉军舰.苹果多酚的组分鉴定及功能特性研究[D].杨凌:西北农林科技大学,2013.