桃果实PpFPPS和PpGGPPS基因的克隆及表达分析
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摘要
本试验以桃果实为试验材料,利用RT-PCR结合RACE技术克隆获得法尼基焦磷酸合成酶(FPPS)和牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)基因的全长c DNA序列,分别命名为Pp FPPS和Pp GGPPS,并对它们进行生物学信息及表达分析。结果表明,Pp FPPS全长1 501 bp,开放阅读框1 029 bp,编码342个氨基酸;Pp GGPPS全长1 547 bp,开放阅读框1 113 bp,编码370个氨基酸。进化树分析表明,Pp FPPS与苹果和梨的亲缘性较高;Pp GGPPS与橡胶树的亲缘性较高。蛋白质序列分析表明,Pp FPPS和Pp GGPPS均含有5个保守域和2个天冬氨酸富集区(FARM和SARM),同属反式-异戊烯转移酶家族。实时荧光定量PCR分析表明,随着果实成熟度的提高,Pp FPPS和Pp GGPPS基因在桃果实果皮和果肉中的表达量总体上呈先增加后降低的趋势,并在膨大和硬熟期达到最大值。果实膨大期后,Pp FPPS和Pp GGPPS基因在果皮中的表达显著高于果肉,这可能是桃果实成熟后期果皮类胡萝卜素含量高于果肉的重要原因。本研究结果为进一步深入研究桃果实类胡萝卜素生物合成的分子机理奠定了理论基础。
Full-length c DNAs of farnesyl pyrophosphate synthase( Pp FPPS) and geranylgeranyl pyrophosphate synthase( Pp GGPPS) were isolated from peach fruit using degenerate RT-PCR and RACE( rapid amplification of c DNA ends)methods,and their biological information and expression were also analyzed. Pp FPPS was 1 501 bp in full length and encoded a predicted protein of 342 amino acids,open reading frame( ORF) length was 1 029 bp. The full length of Pp GGPPS was 1 547 bp with a 1 113 bp ORF,which encoded a deduced polypeptide of 370 amino acids. Phylogenetic analysis indicated that Pp FPPS was similar to FPPS in apple fruit and pear fruit,while Pp GGPPS exhibited identity to the GGPPS of Hevea brasiliensis. Deduced amino acid sequence demonstrated thatboth Pp FPPS and Pp GGPPS proteins contained five conserved domains and two specific Aspartic acid enrichment regions( FARM and SARM),which belonged to the trans-isopentyl transferase family. The quantitative real-time PCR showed that expressionlevel of Pp FPPS and Pp GGPPS in exocarp and mesocarp were increased at the early stage then decreased with the maturity of peach fruit,and reached the maximum at fruit enlarging and maturing period. After fruit enlarging period,the significantly higher level of Pp FPPS and Pp GGPPS transcripts in fruit exocarpwas observed as compared to mesocarp,which may be the major reason that the carotenoid content in fruit exocarp showed higher than in mesocarp. The result lay the foundation for further research of Carotenoid biosynthetic pathway.
Full-length c DNAs of farnesyl pyrophosphate synthase( Pp FPPS) and geranylgeranyl pyrophosphate synthase( Pp GGPPS) were isolated from peach fruit using degenerate RT-PCR and RACE( rapid amplification of c DNA ends)methods,and their biological information and expression were also analyzed. Pp FPPS was 1 501 bp in full length and encoded a predicted protein of 342 amino acids,open reading frame( ORF) length was 1 029 bp. The full length of Pp GGPPS was 1 547 bp with a 1 113 bp ORF,which encoded a deduced polypeptide of 370 amino acids. Phylogenetic analysis indicated that Pp FPPS was similar to FPPS in apple fruit and pear fruit,while Pp GGPPS exhibited identity to the GGPPS of Hevea brasiliensis. Deduced amino acid sequence demonstrated thatboth Pp FPPS and Pp GGPPS proteins contained five conserved domains and two specific Aspartic acid enrichment regions( FARM and SARM),which belonged to the trans-isopentyl transferase family. The quantitative real-time PCR showed that expressionlevel of Pp FPPS and Pp GGPPS in exocarp and mesocarp were increased at the early stage then decreased with the maturity of peach fruit,and reached the maximum at fruit enlarging and maturing period. After fruit enlarging period,the significantly higher level of Pp FPPS and Pp GGPPS transcripts in fruit exocarpwas observed as compared to mesocarp,which may be the major reason that the carotenoid content in fruit exocarp showed higher than in mesocarp. The result lay the foundation for further research of Carotenoid biosynthetic pathway.
引文
[1]张正科.1-MCP与内源乙烯相互作用对番茄和鳄梨成熟生理的影响研究[D].杨凌:西北农林科技大学,2011
[2]陈克明,陈伟,杨震峰.桃果实采后可溶性糖和果胶类物质的变化与低温冷害的关系[J].核农学报,2013,27(5):647-652
[3]Gil M L,Tomás-Barberán F A,Hess-Pierce B,Kader A A.Antioxidant capacities,phenolic compounds,carotenoids,and vitamin c contents of nectarine,peach,and plum cultivars from California[J].Journal of Agricultural and Food Chemistry,2002,50(17):4976-4982
[4]Dalla Valle A Z,Mignani I,Spinardi A,Calvano F,Ciappellano S.The antioxidant profile of three different peaches cultivars(Prunus persica)and their short-term effect on antioxidant status in human[J].European Food Research and Technology,2007,225(2):167-172
[5]周君,陈宗玲,张琼,王红清.套袋对桃果实成熟过程中酚酸类和类黄酮类物质积累的影响[J].园艺学报,2009,36(12):1717-1724
[6]Wright K P,Kader A A.Effect of controlled-atmosphere storage on the quality and carotenoid content of sliced persimmons and peaches[J].Posthavest Biology and Technology,1997,10(1):89-97
[7]Caprioli I,Lafuente M T,Rodrigo M J,Mencarlli F.Influence of postharvest treatments on quality,carotenoids,and abscisic acid content of stored‘Spring Belle’peach(Prunus persica)fruit[J].Journal of Agricultural and Food Chemistry,2009,57(15):7056-7063
[8]梁敏华.桃果实中类胡萝卜素合成基因的克隆及其对蓝光的响应机制研究[D].上海:上海海洋大学,2015
[9]Lichtenthaler H K,Schw ender J,Disch A,Rohmer M.Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate independent pathway[J].FEBS Letters,1997,40(3):271-274
[10]彭刚.柑橘果实转色的叶绿素和类胡萝卜素代谢基础[D].杭州:浙江大学,2013
[11]韦艳萍,庞欣,刘云飞,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,杨悦俭,万红建.植物类胡萝卜素裂解氧化酶研究进展[J].核农学报,2015,28(11):2071-2078
[12]Rodrfguez-Concepcion M,Ahumada I,Diez-Juez E,Sauret-Güeto S,Lois L M,Gallego F,Carretero-Paulet L,Campos N,Boronat A.1-Deoxy-D-xylulose-5-phosphate reducto-isomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[J].The Plant Journal,2001,27(3):213-222
[13]Vandermoten S,Haubruge E,Cusson M.New insightsinto shortchain prenyltransferases:structuralfeatures,evolutionary history and potential for selective inhibition[J].Cellularand Molecular Life Sciences,2009,66(23):3685-3695
[14]李永波,樊庆琦,王宝莲,楚秀生.植物法呢基焦磷酸合酶基因(FPPS)研究进展[J].农业生物技术学报,2012,20(3):321-330
[15]Jones C G,Moniodis J,Zulak K G,Scaffidi A,Plummer J A,Ghisalberti E L,Barbour E L,Bohlmann J.Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase TPS-a and TPS-bsubfamilies,including santalene synthases[J].The Journal of Biological Chemistry,2011,286(20):17445-17454
[16]Kim O T,Bang K H,Jung S J,Kim Y C,Hyun D Y,Kim S H,Cha S W.Molecular characterization of ginsengfarnesyl diphosphate synthase gene and its up-regulation bymethyl jasmonate[J].Biologia Plantarum,2010,54(1):47-53
[17]Okada K,Saito T,Nakagawa T,Kawamukai M,Kamiya Y.Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis[J].Plant Physiology,2000,122(4):1045-1056
[18]Ament K,Van Schie C C,Bouwmeester H J,Schuurink R C.Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of(E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acidand salicylic acid signaling pathways[J].Planta,2006,224(5):1197-1208
[19]Tong Z G,Gao Z H,Wang F.Selection of reliable reference genes for gene expression studies in peach using real-time PCR[J].BMC Molecular Biology,2009,10(1):71
[20]蒙姣荣,陈本勇,黎起秦,李惠,陈保善.罗汉果法呢基焦磷酸合成酶基因的克隆及其序列分析[J].中草药,2011,42(12):2512-2517
[21]Guo R T,Kuo C J,Chou C C,Ko T P,Shr H L,Liang P H,Wang A H.Crystal structure of octaprenyl pyrophosphate synthase from hyperthermophilic Thermotoga maritima and mechanism of product chain length determination[J].Journal of Biological Chemistry,2004,279(6):4903-4912
[22]付楠霞,翟枫,姜鸣,吕舒敏,张雅林.锯角豆芫菁法尼基焦磷酸合酶的c DNA克隆、序列分析及原核表达[J].中国农业科学,2014,47(2):273-283
[23]Sen S E,Trobaugh C,Béliveau C,Richard T,Cusson M.Cloning,expression and characterization of a dipteran farnesyl diphosphatesynthase[J].Insect Biochemistry and Molecular Biology,2007,37(11):1198-1206
[24]Bouvier F,Rahier A,Camara B.Biogenesis,molecular regulationand function of plant isoprenoids[J].Progress in Lipid Research,2005,44(6):357-429
[25]Szkopinska A,Pochocka D.Farnesyl diphosphatesynthase:regulation of product specificity[J].Acta Biochimica Polonica,2005,52(1):45-55
[26]李嵘,王喆之.植物法呢基焦磷酸合成酶的生物信息学分析[J].热带亚热带植物学报,2007,15(2):126-134
[27]Tarshis L C,Proteau P J,Kellogg B A,Sacchettini J C,Poulter C D.Regulation of product chain length byisoprenyl diphosphate synthases[J].Proceedings of the National Academy of Science of the United States of Amercia,1996,93(26):15018-15023
[28]Kim O T,Kim S H,Ohyama K,Muranaka T,Choi Y E,Lee H Y,Kim M Y.Upregulation ofphytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase[J].Plant Cell Reports,2010,29(4):403-411
[29]Sanmiya K,Iwasaki T,Matsuoka M,Miyao M,Yamamoto N.Cloning of a cD NA that encodes farnesyl diphosphate synthase and the blue-light induced expression of the corresponding gene in the leaves of riceplants[J].Biochim Biophy Acta,1997,1350(3):240-246
[30]Laskaris G,De Jong C F,Jaziri M,Van Der Heijden R,Theodoridis G,Verpoorte R.Geranylgeranyl diphosphate synthase activity and taxane production in Taxus baccata cells[J].Phytochemistry,1999,50(6):939-946
[31]李翠萍.LeG GPS2,AtC AO与AtH EMA1基因对烟草耐弱光性的影响[D].重庆:西南大学,2013
[2]陈克明,陈伟,杨震峰.桃果实采后可溶性糖和果胶类物质的变化与低温冷害的关系[J].核农学报,2013,27(5):647-652
[3]Gil M L,Tomás-Barberán F A,Hess-Pierce B,Kader A A.Antioxidant capacities,phenolic compounds,carotenoids,and vitamin c contents of nectarine,peach,and plum cultivars from California[J].Journal of Agricultural and Food Chemistry,2002,50(17):4976-4982
[4]Dalla Valle A Z,Mignani I,Spinardi A,Calvano F,Ciappellano S.The antioxidant profile of three different peaches cultivars(Prunus persica)and their short-term effect on antioxidant status in human[J].European Food Research and Technology,2007,225(2):167-172
[5]周君,陈宗玲,张琼,王红清.套袋对桃果实成熟过程中酚酸类和类黄酮类物质积累的影响[J].园艺学报,2009,36(12):1717-1724
[6]Wright K P,Kader A A.Effect of controlled-atmosphere storage on the quality and carotenoid content of sliced persimmons and peaches[J].Posthavest Biology and Technology,1997,10(1):89-97
[7]Caprioli I,Lafuente M T,Rodrigo M J,Mencarlli F.Influence of postharvest treatments on quality,carotenoids,and abscisic acid content of stored‘Spring Belle’peach(Prunus persica)fruit[J].Journal of Agricultural and Food Chemistry,2009,57(15):7056-7063
[8]梁敏华.桃果实中类胡萝卜素合成基因的克隆及其对蓝光的响应机制研究[D].上海:上海海洋大学,2015
[9]Lichtenthaler H K,Schw ender J,Disch A,Rohmer M.Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate independent pathway[J].FEBS Letters,1997,40(3):271-274
[10]彭刚.柑橘果实转色的叶绿素和类胡萝卜素代谢基础[D].杭州:浙江大学,2013
[11]韦艳萍,庞欣,刘云飞,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,杨悦俭,万红建.植物类胡萝卜素裂解氧化酶研究进展[J].核农学报,2015,28(11):2071-2078
[12]Rodrfguez-Concepcion M,Ahumada I,Diez-Juez E,Sauret-Güeto S,Lois L M,Gallego F,Carretero-Paulet L,Campos N,Boronat A.1-Deoxy-D-xylulose-5-phosphate reducto-isomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[J].The Plant Journal,2001,27(3):213-222
[13]Vandermoten S,Haubruge E,Cusson M.New insightsinto shortchain prenyltransferases:structuralfeatures,evolutionary history and potential for selective inhibition[J].Cellularand Molecular Life Sciences,2009,66(23):3685-3695
[14]李永波,樊庆琦,王宝莲,楚秀生.植物法呢基焦磷酸合酶基因(FPPS)研究进展[J].农业生物技术学报,2012,20(3):321-330
[15]Jones C G,Moniodis J,Zulak K G,Scaffidi A,Plummer J A,Ghisalberti E L,Barbour E L,Bohlmann J.Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase TPS-a and TPS-bsubfamilies,including santalene synthases[J].The Journal of Biological Chemistry,2011,286(20):17445-17454
[16]Kim O T,Bang K H,Jung S J,Kim Y C,Hyun D Y,Kim S H,Cha S W.Molecular characterization of ginsengfarnesyl diphosphate synthase gene and its up-regulation bymethyl jasmonate[J].Biologia Plantarum,2010,54(1):47-53
[17]Okada K,Saito T,Nakagawa T,Kawamukai M,Kamiya Y.Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis[J].Plant Physiology,2000,122(4):1045-1056
[18]Ament K,Van Schie C C,Bouwmeester H J,Schuurink R C.Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of(E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acidand salicylic acid signaling pathways[J].Planta,2006,224(5):1197-1208
[19]Tong Z G,Gao Z H,Wang F.Selection of reliable reference genes for gene expression studies in peach using real-time PCR[J].BMC Molecular Biology,2009,10(1):71
[20]蒙姣荣,陈本勇,黎起秦,李惠,陈保善.罗汉果法呢基焦磷酸合成酶基因的克隆及其序列分析[J].中草药,2011,42(12):2512-2517
[21]Guo R T,Kuo C J,Chou C C,Ko T P,Shr H L,Liang P H,Wang A H.Crystal structure of octaprenyl pyrophosphate synthase from hyperthermophilic Thermotoga maritima and mechanism of product chain length determination[J].Journal of Biological Chemistry,2004,279(6):4903-4912
[22]付楠霞,翟枫,姜鸣,吕舒敏,张雅林.锯角豆芫菁法尼基焦磷酸合酶的c DNA克隆、序列分析及原核表达[J].中国农业科学,2014,47(2):273-283
[23]Sen S E,Trobaugh C,Béliveau C,Richard T,Cusson M.Cloning,expression and characterization of a dipteran farnesyl diphosphatesynthase[J].Insect Biochemistry and Molecular Biology,2007,37(11):1198-1206
[24]Bouvier F,Rahier A,Camara B.Biogenesis,molecular regulationand function of plant isoprenoids[J].Progress in Lipid Research,2005,44(6):357-429
[25]Szkopinska A,Pochocka D.Farnesyl diphosphatesynthase:regulation of product specificity[J].Acta Biochimica Polonica,2005,52(1):45-55
[26]李嵘,王喆之.植物法呢基焦磷酸合成酶的生物信息学分析[J].热带亚热带植物学报,2007,15(2):126-134
[27]Tarshis L C,Proteau P J,Kellogg B A,Sacchettini J C,Poulter C D.Regulation of product chain length byisoprenyl diphosphate synthases[J].Proceedings of the National Academy of Science of the United States of Amercia,1996,93(26):15018-15023
[28]Kim O T,Kim S H,Ohyama K,Muranaka T,Choi Y E,Lee H Y,Kim M Y.Upregulation ofphytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase[J].Plant Cell Reports,2010,29(4):403-411
[29]Sanmiya K,Iwasaki T,Matsuoka M,Miyao M,Yamamoto N.Cloning of a cD NA that encodes farnesyl diphosphate synthase and the blue-light induced expression of the corresponding gene in the leaves of riceplants[J].Biochim Biophy Acta,1997,1350(3):240-246
[30]Laskaris G,De Jong C F,Jaziri M,Van Der Heijden R,Theodoridis G,Verpoorte R.Geranylgeranyl diphosphate synthase activity and taxane production in Taxus baccata cells[J].Phytochemistry,1999,50(6):939-946
[31]李翠萍.LeG GPS2,AtC AO与AtH EMA1基因对烟草耐弱光性的影响[D].重庆:西南大学,2013