红肉火龙果(Hylocereus polyrhizus)果实与茎转录组测序分析
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摘要
为探讨红肉火龙果果实和茎转录组模式及可溶性糖和甜菜苷色素积累的分子基础,采用Illumina HiSeq 4000技术对果实果肉和茎进行转录组测序。共获得28.81 Gb的Clean read,组装获得79 658个Unigene(54.98 Mb),平均长度为690 bp。35 139个Unigene能够在NR、NT、Swiss-Prot、KEGG、COG、PFAM和GO数据库中注释。大量Unigene参与多条代谢途径,包括碳水化合物和氨基酸代谢、次生代谢、柠檬酸循环、果糖和甘露糖代谢等。在果实果肉中上调表达的Unigene主要参与淀粉、蔗糖代谢途径和谷胱甘肽代谢,在茎中上调表达的Unigene主要参与碳代谢和光合作用代谢。筛选获得5个与果实可溶性糖和甜菜苷色素积累的重要候选Unigene。本研究结果克隆的火龙果果实可溶性糖和甜菜苷色素积累相关功能基因,对探讨基因的生理功能和调控机制,改良火龙果果实品质提供重要基础。
In order to investi gate transcriptional patterns of the fruit and stem, and molecular basis of soluble sugar and betanin pigment accumulation, the fruit flesh and stem were sequenced the transcriptome using Illumina Hi Seq 4000 technique. A total of 28.81 Gb of clean read was obtained, and 79 658 unigene(54.98 Mb) were generated with an average length of 690 bp. 35 139 unigene could be annotated on NR, NT, Swiss-Prot, KEGG,COG, PFAM and GO databases. A large number of unigene were involved in multiple metabolic pathways,including carbohydrate and amino acid metabolism, secondary metabolism, citric acid cycle, fructose and mannose metabolism. The up-regulated expression of unigenes in fruit fresh mainly involved in starch, sucrose metabolic pathways and glutathione metabolism, and the up-regulated expression of unigenes in stem mainly involved in carbon metabolism and photosynthetic metabolism. Five candidate functional unigenes involved in soluble sugar and betanin pigments accumulation in fruits were obtained by screening. The result of this study might provide an important basis for cloning functional genes related to the soluble sugar and betanin pigments accumulation in pitaya fruits, exploring the physiological function and regulation mechanism of genes, and improving fruit quality.
In order to investi gate transcriptional patterns of the fruit and stem, and molecular basis of soluble sugar and betanin pigment accumulation, the fruit flesh and stem were sequenced the transcriptome using Illumina Hi Seq 4000 technique. A total of 28.81 Gb of clean read was obtained, and 79 658 unigene(54.98 Mb) were generated with an average length of 690 bp. 35 139 unigene could be annotated on NR, NT, Swiss-Prot, KEGG,COG, PFAM and GO databases. A large number of unigene were involved in multiple metabolic pathways,including carbohydrate and amino acid metabolism, secondary metabolism, citric acid cycle, fructose and mannose metabolism. The up-regulated expression of unigenes in fruit fresh mainly involved in starch, sucrose metabolic pathways and glutathione metabolism, and the up-regulated expression of unigenes in stem mainly involved in carbon metabolism and photosynthetic metabolism. Five candidate functional unigenes involved in soluble sugar and betanin pigments accumulation in fruits were obtained by screening. The result of this study might provide an important basis for cloning functional genes related to the soluble sugar and betanin pigments accumulation in pitaya fruits, exploring the physiological function and regulation mechanism of genes, and improving fruit quality.
引文
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Chardon F.,Bedu M.,Calenge F.,Klemens P.A.,Spinner L.,Clement G.,Chietera G.,Léran S.,Ferrand M.,Lacombe B.,Loudet O.,Dinant S.,Bellini C.,Neuhaus H.E.,DanielVedele F.,and Krapp A.,2013,Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis,Curr.Biol.,23(8):697-702
Christinet L.,Burdet F.X.,Zaiko M.,Hinz U.,and Zryd J.P.,2004,Characterization and functional identification of a novel plant 4,5-Extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora,Plant Physiol.,134(1):265-274
Cohen H.,Fait A.,and Tel-Zur N.,2013,Morphological,cytological and metabolic consequences of autopolyploidization in Hylocereus(Cactaceae)species,BMC Plant Biol.,13(1):173
Du J.,Wang S.D.,He C.M.,Zhou B.,Ruan Y.L.,and Shou H.X.,2017,Identification of regulatory networks and hub genes controlling soybean seed set and size using RNA sequencing analysis,J.Exp.Bot.,68(8):1955-1972
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Guo W.J.,Nagy R.,Chen H.Y.,Pfrunder S.,Yu Y.C.,Santelia D.,Frommer W.B.,and Martinoia E.,2014,SWEET17,a facilitative transporter,mediates fructose transport across the tonoplast of Arabidopsis roots and leaves,Plant Physiol.,164(2):777-789
Hatlestad G.J.,Sunnadeniya R.M.,Akhavan N.A.,Gonzalez A.,Goldman I.L.,McGrath J.M.,and Lloyd A.M.,2012,The beet R locus encodes a new cytochrome P450 required for red betalain production,Nat.Genet.,44(7):816-820
Jiang F.X.,Jiang X.Y.,Wu S.,Yang L.J.,and Chen Q.B.,2015Transcriptome sequencing and characteristic analysis of Ornithogalum caudatum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(9):2038-2050(姜福星,江欣燕,吴生杨丽娟,陈其兵,2015,虎眼万年青鳞茎转录组特性分析分子植物育种,13(9):2038-2050)
Jin J.J.,Sun Y.W.,Qu J.,syah R.,Lim C.H.,Alfiko Y.,Rahman N.E.B.,Suwanto A.,Yue G.H.,Wong L.,Chua N.H.,and Jian Y.,2017,Transcriptome and functional analysis reveals hybrid vigor for oil biosynthesis in oil palm,Sci.Rep.,7(1)439
Jung B.,Ludewig F.,Schulz A.,Meiner G.,Wostefeld N.,Flügge U.I.,Pommerrenig B.,Wirsching P.,Sauer N.,Koch W.Sommer F.,Mühlhaus T.,Schroda M.,Cuin T.A.,Graus D.Marten I.,Hedrich R.,and Neuhaus H.E.,2015,Identification of the transporter responsible for sucrose accumulation in sugar beet taproots,Nat.Plants,1(1):14001
Katz M.,Fon Y.J.,Lee B.S.,Phinney A.,Sadka A.,and Blumwald E.,2007,The citrus fruit proteome:insights into citrus frui metabolism,Planta,226(4):989-1005
Li W.Y.,Peng Z.J.,Wang B.,and Cai Y.Q.,2010,Content and composition of soluble sugar and organic acid in flesh of different pitaya varieties,Guizhou Nongye Kexue(Guizhou Agriculture Sciences),38(11):215-217(李文云,彭志军,王彬,蔡永强,2010,火龙果不同品种(品系)果肉糖,酸含量及组成分析,贵州农业科学,38(11):215-217)
Liu J.,Luo C.,Sun W.,and Yi Y.,2017,Transcriptomics analysis of hard seeds of Sophora viciifolia,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):867-874(刘杰,罗充,孙威,乙引,2017,白刺花种子转录组分析,分子植物育种15(3):867-874)
Nurul S.R.,and Asmah R.,2014,Variability in nutritional composition and phytochemical properties of red pitaya(Hylocereus polyrhizus)from Malaysia and Australia,International Food Research Journal,21(4):1689-1697
Ruan Y.L.,2014,Sucrose metabolism:gateway to diverse carbon use and sugar signaling,Annu.Rev.Plant Biol.,65(1):33-67
Sasaki N.,Abe Y.,Goda Y.,Adachi T.,Kasahara K.,and Ozek Y.,2009,Detection of DOPA 4,5-Dioxygenase(DOD)activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa,Plant Cell Physiol.,50(5):1012-1016
Schulz A.,Beyhl D.,Marten I.,Wormit A.,Neuhaus E.,Poschet G.,Büttner M.,Schneider S.,Sauer N.,and Hedrich R.,2011,Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2,Plant J.,68:129-136
Stintzing F.C.,Schieber A.,and Carle R.,2002,Identification of betalains from yellow beet(Beta vulgaris L.)and cactus pear[Opuntia ficus-indica(L.)Mill.]by high-performance liquid chromatography-electrospray ionization mass spectrometry,J.Agri.Food Chem.,50(8):2302-2307
Tanaka Y.,Sasaki N.,and Ohmiya A.,2008,Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids,Plant J.,54(4):733-749
Wang G.H.,Zhao C.,Zhang M.,Li Y.Z.,Shen Y.Y.,and Guo J.X.,2017,Transcriptome analysis around the onset of strawberry fruit ripening uncovers an important role of oxidative phosphorylation in ripening,Sci.Rep.,7:41477
Wang Y.P.,Chen J.W.,Feng J.J.,Qin Q.P.,and Huang J.P.,2015,Overexpression of a loquat(Eriobotrya japonica Lind.)vacuolar invertase affects sucrose levels and growth,Plant Cell Tissue and Organ Culture,123(1):99-108
Wingenter K.,Schulz A.,Wormit A.,Trentmann O.,Wic S.,Hoermiller,Heyer A.G.,Marten,Hedrich R.,and Neuhaus H.E.,2010,Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis,Plant Physiol.,154(2):665-677
Wu C.,Peng J.,Xiang L.,Gu M.,and Guo F.Q.,2016,Sequencing and analysis of the transcriptome of Dendrobium officinale,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3334-3346(吴超,彭娟,向林,顾敏,郭方其,2016,基于高通量测序的铁皮石斛叶片转录组分析,分子植物育种,14(12):3334-3346)
Zhao Y.C.,Liu X.W.,Li Y.,and Zhao D.G.,2016,Transcriptome data assembly and gene Function annotation of Zanthoxylum piperitum DC var.inerme makino,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1805-1819(赵懿琛,刘雪微,李岩,赵德刚,2016,朝仓花椒幼芽转录组测序数据组装及基因功能注释,基因组学与应用生物学,35(7):1805-1819)
Chardon F.,Bedu M.,Calenge F.,Klemens P.A.,Spinner L.,Clement G.,Chietera G.,Léran S.,Ferrand M.,Lacombe B.,Loudet O.,Dinant S.,Bellini C.,Neuhaus H.E.,DanielVedele F.,and Krapp A.,2013,Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis,Curr.Biol.,23(8):697-702
Christinet L.,Burdet F.X.,Zaiko M.,Hinz U.,and Zryd J.P.,2004,Characterization and functional identification of a novel plant 4,5-Extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora,Plant Physiol.,134(1):265-274
Cohen H.,Fait A.,and Tel-Zur N.,2013,Morphological,cytological and metabolic consequences of autopolyploidization in Hylocereus(Cactaceae)species,BMC Plant Biol.,13(1):173
Du J.,Wang S.D.,He C.M.,Zhou B.,Ruan Y.L.,and Shou H.X.,2017,Identification of regulatory networks and hub genes controlling soybean seed set and size using RNA sequencing analysis,J.Exp.Bot.,68(8):1955-1972
Gandaherrero F.,and Garcacarmona F.,2013,Biosynthesis of betalains:yellow and violet plant pigments,Trends Plant Sci.,18(6):334-343
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.,Chen Z.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,di Palma F.,Birren B.W.,Nusbaum C.,Lindblad-Toh K.,Friedman N.,and Regev A.,2011,Full-length transcriptome assembly from RNA-Seq data without a reference genome,Nat.Biotechnol.,29(7):644-652
Guo W.J.,Nagy R.,Chen H.Y.,Pfrunder S.,Yu Y.C.,Santelia D.,Frommer W.B.,and Martinoia E.,2014,SWEET17,a facilitative transporter,mediates fructose transport across the tonoplast of Arabidopsis roots and leaves,Plant Physiol.,164(2):777-789
Hatlestad G.J.,Sunnadeniya R.M.,Akhavan N.A.,Gonzalez A.,Goldman I.L.,McGrath J.M.,and Lloyd A.M.,2012,The beet R locus encodes a new cytochrome P450 required for red betalain production,Nat.Genet.,44(7):816-820
Jiang F.X.,Jiang X.Y.,Wu S.,Yang L.J.,and Chen Q.B.,2015Transcriptome sequencing and characteristic analysis of Ornithogalum caudatum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(9):2038-2050(姜福星,江欣燕,吴生杨丽娟,陈其兵,2015,虎眼万年青鳞茎转录组特性分析分子植物育种,13(9):2038-2050)
Jin J.J.,Sun Y.W.,Qu J.,syah R.,Lim C.H.,Alfiko Y.,Rahman N.E.B.,Suwanto A.,Yue G.H.,Wong L.,Chua N.H.,and Jian Y.,2017,Transcriptome and functional analysis reveals hybrid vigor for oil biosynthesis in oil palm,Sci.Rep.,7(1)439
Jung B.,Ludewig F.,Schulz A.,Meiner G.,Wostefeld N.,Flügge U.I.,Pommerrenig B.,Wirsching P.,Sauer N.,Koch W.Sommer F.,Mühlhaus T.,Schroda M.,Cuin T.A.,Graus D.Marten I.,Hedrich R.,and Neuhaus H.E.,2015,Identification of the transporter responsible for sucrose accumulation in sugar beet taproots,Nat.Plants,1(1):14001
Katz M.,Fon Y.J.,Lee B.S.,Phinney A.,Sadka A.,and Blumwald E.,2007,The citrus fruit proteome:insights into citrus frui metabolism,Planta,226(4):989-1005
Li W.Y.,Peng Z.J.,Wang B.,and Cai Y.Q.,2010,Content and composition of soluble sugar and organic acid in flesh of different pitaya varieties,Guizhou Nongye Kexue(Guizhou Agriculture Sciences),38(11):215-217(李文云,彭志军,王彬,蔡永强,2010,火龙果不同品种(品系)果肉糖,酸含量及组成分析,贵州农业科学,38(11):215-217)
Liu J.,Luo C.,Sun W.,and Yi Y.,2017,Transcriptomics analysis of hard seeds of Sophora viciifolia,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):867-874(刘杰,罗充,孙威,乙引,2017,白刺花种子转录组分析,分子植物育种15(3):867-874)
Nurul S.R.,and Asmah R.,2014,Variability in nutritional composition and phytochemical properties of red pitaya(Hylocereus polyrhizus)from Malaysia and Australia,International Food Research Journal,21(4):1689-1697
Ruan Y.L.,2014,Sucrose metabolism:gateway to diverse carbon use and sugar signaling,Annu.Rev.Plant Biol.,65(1):33-67
Sasaki N.,Abe Y.,Goda Y.,Adachi T.,Kasahara K.,and Ozek Y.,2009,Detection of DOPA 4,5-Dioxygenase(DOD)activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa,Plant Cell Physiol.,50(5):1012-1016
Schulz A.,Beyhl D.,Marten I.,Wormit A.,Neuhaus E.,Poschet G.,Büttner M.,Schneider S.,Sauer N.,and Hedrich R.,2011,Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2,Plant J.,68:129-136
Stintzing F.C.,Schieber A.,and Carle R.,2002,Identification of betalains from yellow beet(Beta vulgaris L.)and cactus pear[Opuntia ficus-indica(L.)Mill.]by high-performance liquid chromatography-electrospray ionization mass spectrometry,J.Agri.Food Chem.,50(8):2302-2307
Tanaka Y.,Sasaki N.,and Ohmiya A.,2008,Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids,Plant J.,54(4):733-749
Wang G.H.,Zhao C.,Zhang M.,Li Y.Z.,Shen Y.Y.,and Guo J.X.,2017,Transcriptome analysis around the onset of strawberry fruit ripening uncovers an important role of oxidative phosphorylation in ripening,Sci.Rep.,7:41477
Wang Y.P.,Chen J.W.,Feng J.J.,Qin Q.P.,and Huang J.P.,2015,Overexpression of a loquat(Eriobotrya japonica Lind.)vacuolar invertase affects sucrose levels and growth,Plant Cell Tissue and Organ Culture,123(1):99-108
Wingenter K.,Schulz A.,Wormit A.,Trentmann O.,Wic S.,Hoermiller,Heyer A.G.,Marten,Hedrich R.,and Neuhaus H.E.,2010,Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning,sugar signaling,and seed yield in Arabidopsis,Plant Physiol.,154(2):665-677
Wu C.,Peng J.,Xiang L.,Gu M.,and Guo F.Q.,2016,Sequencing and analysis of the transcriptome of Dendrobium officinale,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3334-3346(吴超,彭娟,向林,顾敏,郭方其,2016,基于高通量测序的铁皮石斛叶片转录组分析,分子植物育种,14(12):3334-3346)
Zhao Y.C.,Liu X.W.,Li Y.,and Zhao D.G.,2016,Transcriptome data assembly and gene Function annotation of Zanthoxylum piperitum DC var.inerme makino,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1805-1819(赵懿琛,刘雪微,李岩,赵德刚,2016,朝仓花椒幼芽转录组测序数据组装及基因功能注释,基因组学与应用生物学,35(7):1805-1819)