皂荚类胡萝卜素合成途径基因表达分析及miRNA调控的鉴定
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摘要
皂荚是豆科皂荚属落叶乔木,具有较高的生态价值和药用价值。研究相关基因的表达、调控及功能,是分子改良植物的重要前提,而目前对皂荚基因表达调控的研究鲜有报道。为了探究皂荚荚果成熟过程中类胡萝卜素合成途径的基因表达和调控,本研究基于皂荚荚果两个不同发育期(7月, 9月)的转录组高通量测序结果,发现合成类胡萝卜素关键前体番茄红素的合成酶基因(CrtQ)在皂荚荚果成熟早期被显著抑制,而负责内源激素脱落酸(ABA)的合成酶基因(AAO3)被显著诱导;另外,研究发现miR838-3p可调控类胡萝卜素途径中合成番茄红素的前体的酶基因(CrtB)。本研究结果表明,类胡萝卜素途径中色素合成过程被miR838-3p抑制进而诱导脱落酸的合成从而在皂荚荚果成熟中起着重要作用,并可能会反馈促进次生代谢物皂苷的生成。研究首次鉴定了皂荚荚果发育中类胡萝卜素合成途径基因的表达模式,并揭示了miRNA的调控,该结果可为提高皂荚抗逆性或遗传改良荚果内重要次生代谢物皂苷含量提供分子线索和依据。
Gleditsia sinensis Lam. is a kind of d eciduous tree of leguminous Gleditschia L., which with high ecological and medicinal value. It is a highly important prerequisite to investigate the differential expression regulation of related genes and elucidate the genetic function for the molecular improvement of plants. However,so far there are few reports on the gene expression and regulation of G. sinensis. In order to investigate the expression and regulation of genes involved in carotenoid synthesis pathway during the development process of G.sinensis pods ripening, this study was based on high-throughput transcriptome sequencing results of G. sinensis pods from two different development stages(July and September). It was found that the synthase gene(CrtQ) of lycopene, the key precursor of carotenoid was significantly inhibited during early ripening. However, the synthase gene(AAO3) responsible for endogenous hormone abscisic acid(ABA) was were significantly induced. Interestingly,miR838-3 p was found to regulate the enzyme genes CrtB of the precursor of lycopene synthesis in the carotenoid pathway. The results of this study indicated that the in the carotenoid pathway, the pigment synthesis process was inhibited by mir838-3 p, and then the synthesis of abscisic acid was induced, thus playing an important role in the maturation of acacia pod. There might also have a feedback to promote the production of secondary metabolite saponin. This is the first report on the expression pattern of genes involved in carotenoid biosynthesis pathway, and it uncovered the involvement of miRNAs in the regulation. Combinedly, this investigation might provide molecular clues and basis for improving the anti-inversion of saponids or genetically improving the content of important secondary metabolites in G. sinensis pods.
Gleditsia sinensis Lam. is a kind of d eciduous tree of leguminous Gleditschia L., which with high ecological and medicinal value. It is a highly important prerequisite to investigate the differential expression regulation of related genes and elucidate the genetic function for the molecular improvement of plants. However,so far there are few reports on the gene expression and regulation of G. sinensis. In order to investigate the expression and regulation of genes involved in carotenoid synthesis pathway during the development process of G.sinensis pods ripening, this study was based on high-throughput transcriptome sequencing results of G. sinensis pods from two different development stages(July and September). It was found that the synthase gene(CrtQ) of lycopene, the key precursor of carotenoid was significantly inhibited during early ripening. However, the synthase gene(AAO3) responsible for endogenous hormone abscisic acid(ABA) was were significantly induced. Interestingly,miR838-3 p was found to regulate the enzyme genes CrtB of the precursor of lycopene synthesis in the carotenoid pathway. The results of this study indicated that the in the carotenoid pathway, the pigment synthesis process was inhibited by mir838-3 p, and then the synthesis of abscisic acid was induced, thus playing an important role in the maturation of acacia pod. There might also have a feedback to promote the production of secondary metabolite saponin. This is the first report on the expression pattern of genes involved in carotenoid biosynthesis pathway, and it uncovered the involvement of miRNAs in the regulation. Combinedly, this investigation might provide molecular clues and basis for improving the anti-inversion of saponids or genetically improving the content of important secondary metabolites in G. sinensis pods.
引文
Audic S.,and Claverie J.M.,1997,The significance of digital gene expression profiles,Genome Res.,7(10):986-995
Chahtane H.,Kim W.,and Lopez-Molina L.,2017,Primary seed dormancy:a temporally multilayered riddle waiting to be unlocked,J.Exp.Bot.,68(4):857-869
Dai X.,and Zhao P.X.,2011,psRNATarget:a plant small RNAtarget analysis server,Nucleic Acids Res.,39:155-159
Dhar M.K.,Sharma R.,Koul A.,and Kaul S.,2015,Development of fruit color in Solanaceae:a story of two biosynthetic pathways,Brief.Funct.Genomics.,14(3):199-212
Feng C.,Tang H.R.,Jiang L.Y.,Wang X.R.,Chen Q.,and Sun B.,2017,Advances in studies on carotenoid in Malus pumila,Zhiwu Kexue Xuebao(Plant Science Journal),35(6):932-939(冯琛,汤浩茹,江雷雨,王小蓉,陈清,孙勃,2017,苹果类胡萝卜素研究进展,植物科学学报,35(6):932-939)
Feng H.,Yi S.L.,Zuo J.Q.,Xie J.H.,and Huang X.,2014,Progress of flower colour modification by genetic engineering and anthocyanin biosynthesis pathway,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):445-451(冯欢,易姝利,左佳琦,谢佳恒,黄萱,2014,基因工程及花色素合成途径在花色改良中的研究进展,基因组学与应用生物学,33(2):445-451)
Frankowski K.,Wilmowicz E.,Kucko A.,Sidlowska M.,Kesy J.,and Kopcewicz J.,2013,Abscisic acid metabolism,Postepy Biochem.,59(1):83-88
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma Z.,Birren B.W.,Nusbaum C.,Lindblad-Toh K.,Friedman N.,and Regev A.,2011,Trinity reconstructing a full-length transcriptome without a genome from RNA-Seq data,Nat.Biotechnol.,29(7):644-652
Guo Y.,and Wang T.M.,2016,The down regulated genes related with creeping-rooted characteristics of alfalfa based on the RNA-seq,Caoye Kexue(Pratacultural Science),33(1):75-85(郭云,王铁梅,2016,基于RNA-Seq技术的苜蓿根蘖性状发生相关下调基因,草业科学,33(1):75-85)
Han S.,Wu Z.,Wang X.,Huang K.,Jin Y.,Yang W.,and Shi H.,2016,De novo assembly and characterization of Gleditsia sinensis transcriptome and subsequent gene identification and SSR mining,Genet.Mol.Res.,15(1):gmr.15017740
Hashimoto H.,Uragami C.,and Cogdell R.J.,2016,Carotenoids and photosynthesis,Subcell.Biochem.,79:111-139
Hauser F.,Li Z.,Waadt R.,and Schroeder J.I.,2017,SnapShot:abscisic acid signaling,Cell,171(7):1708
Hou J.,Sun F.S.,Wu Q.M.,Yang Y.Q.,He W.,and Wang Y.W.,2014,An efficient method for total RNA extraction of poplar bark infected with pathogen and the application,Zhiwu Shengli Xuebao(Plant Physiology Journal),50(2):223-228(侯佳,孙丰硕,吴秋明,杨玉巧,贺伟,王延伟,2014,一种高效提取杨树发病树皮总RNA的方法及应用,植物生理学报,50(2):223-228)
Humplik J.F.,Bergougnoux V.,and Van V.E.,2017,To stimulate or inhibit?That is the question for the function of abscisic acid,Trends Plant Sci.,22(10):830-841
Kanehisa M.,Araki M.,Goto S.,Hattori M.,Hirakawa M.,Itoh M.,Katayama T.,Kawashima S.,Okuda S.,Tokimatsu T.,and Yamanishi Y.,2008,KEGG for linking genomes to life and the environment,Nucleic Acids Res.,36(D):480-484
Khoo H.E.,Prasad K.N.,Kong K.W.,Jiang Y.,and Ismail A.,2011,Carotenoids and their isomers:color pigments in fruits and vegetables,Molecules,16(2):1710-1738
Liang J.Y.,An X.N.,Jiang J.X.,Zhu L.W.,and Zhang W.M.,2003,Study on the chemical constituents of the pod of Gleditsia sinensis,Zhongguo Yesheng Zhiwu Ziyuan(Chinese Wild Plant Resources),22(3):44-46(梁静谊,安鑫南,蒋建新,朱莉伟,张卫明,2003,皂荚化学组成的研究,中国野生植物资源,22(3):44-46)
Liu X.H.,Sun H.Y.,Chen W.W.,Guo Q.G.,Li X.L.,and Liang G.L.,2016,Advances in studies on carotenoids in loquat fruit,Guoshu Xuebao(Journal of Fruit Science),33(7):874-881(刘小洪,孙海艳,陈薇薇,郭启高,李晓林,梁国鲁,2016,枇杷果实类胡萝卜素研究进展,果树学报,33(7):874-881)
Mortazavi A.,Williams B.A.,McCue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nat.Methods,5(7):621-628
Sandmann G.,2015,Carotenoids of biotechnological importance,Adv.Biochem.Eng.Biotechnol.,148:449-467
Yin T.M.,2010,A review of the genomic and gene cloning studies in trees,Yichuan(Hereditas),32(7):677-684(尹佟明,2010,林木基因组及功能基因克隆研究概述,遗传,32(7):677-684)
Yue J.,Lu X.,Zhang H.,Ge J.,Gao X.,and Liu Y.,2017,Identification of conserved and novel microRNAs in blueberry,Front.Plant Sci.,8:1155
Zhang J.P.,Tian X.H.,Yang Y.X.,Liu Q.X.,Wang Q.,Chen L.P.,Li H.L.,and Zhang W.D.,2016,Gleditsia species:an ethnomedical,phytochemical and pharmacological review,J.Ethnopharmacol.,178:155-171
Zhang X.,Zheng Y.,Jagadeeswaran G.,Ren R.,Sunkar R.,and Jiang H.,2014,Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes,Insect Biochem.Mol.Biol.,47:12-22
Zhu J.,Chai L.,Hou B.Z.,Shen Y.Y.,and Guo J.X.,2015,Research advances and prospects of ABA in fleshy fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),42(9):1664-1672(祝军,柴璐,侯柄竹,沈元月,郭家选,2015,果实中脱落酸的研究进展与展望,园艺学报,42(9):1664-1672)
Chahtane H.,Kim W.,and Lopez-Molina L.,2017,Primary seed dormancy:a temporally multilayered riddle waiting to be unlocked,J.Exp.Bot.,68(4):857-869
Dai X.,and Zhao P.X.,2011,psRNATarget:a plant small RNAtarget analysis server,Nucleic Acids Res.,39:155-159
Dhar M.K.,Sharma R.,Koul A.,and Kaul S.,2015,Development of fruit color in Solanaceae:a story of two biosynthetic pathways,Brief.Funct.Genomics.,14(3):199-212
Feng C.,Tang H.R.,Jiang L.Y.,Wang X.R.,Chen Q.,and Sun B.,2017,Advances in studies on carotenoid in Malus pumila,Zhiwu Kexue Xuebao(Plant Science Journal),35(6):932-939(冯琛,汤浩茹,江雷雨,王小蓉,陈清,孙勃,2017,苹果类胡萝卜素研究进展,植物科学学报,35(6):932-939)
Feng H.,Yi S.L.,Zuo J.Q.,Xie J.H.,and Huang X.,2014,Progress of flower colour modification by genetic engineering and anthocyanin biosynthesis pathway,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):445-451(冯欢,易姝利,左佳琦,谢佳恒,黄萱,2014,基因工程及花色素合成途径在花色改良中的研究进展,基因组学与应用生物学,33(2):445-451)
Frankowski K.,Wilmowicz E.,Kucko A.,Sidlowska M.,Kesy J.,and Kopcewicz J.,2013,Abscisic acid metabolism,Postepy Biochem.,59(1):83-88
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma Z.,Birren B.W.,Nusbaum C.,Lindblad-Toh K.,Friedman N.,and Regev A.,2011,Trinity reconstructing a full-length transcriptome without a genome from RNA-Seq data,Nat.Biotechnol.,29(7):644-652
Guo Y.,and Wang T.M.,2016,The down regulated genes related with creeping-rooted characteristics of alfalfa based on the RNA-seq,Caoye Kexue(Pratacultural Science),33(1):75-85(郭云,王铁梅,2016,基于RNA-Seq技术的苜蓿根蘖性状发生相关下调基因,草业科学,33(1):75-85)
Han S.,Wu Z.,Wang X.,Huang K.,Jin Y.,Yang W.,and Shi H.,2016,De novo assembly and characterization of Gleditsia sinensis transcriptome and subsequent gene identification and SSR mining,Genet.Mol.Res.,15(1):gmr.15017740
Hashimoto H.,Uragami C.,and Cogdell R.J.,2016,Carotenoids and photosynthesis,Subcell.Biochem.,79:111-139
Hauser F.,Li Z.,Waadt R.,and Schroeder J.I.,2017,SnapShot:abscisic acid signaling,Cell,171(7):1708
Hou J.,Sun F.S.,Wu Q.M.,Yang Y.Q.,He W.,and Wang Y.W.,2014,An efficient method for total RNA extraction of poplar bark infected with pathogen and the application,Zhiwu Shengli Xuebao(Plant Physiology Journal),50(2):223-228(侯佳,孙丰硕,吴秋明,杨玉巧,贺伟,王延伟,2014,一种高效提取杨树发病树皮总RNA的方法及应用,植物生理学报,50(2):223-228)
Humplik J.F.,Bergougnoux V.,and Van V.E.,2017,To stimulate or inhibit?That is the question for the function of abscisic acid,Trends Plant Sci.,22(10):830-841
Kanehisa M.,Araki M.,Goto S.,Hattori M.,Hirakawa M.,Itoh M.,Katayama T.,Kawashima S.,Okuda S.,Tokimatsu T.,and Yamanishi Y.,2008,KEGG for linking genomes to life and the environment,Nucleic Acids Res.,36(D):480-484
Khoo H.E.,Prasad K.N.,Kong K.W.,Jiang Y.,and Ismail A.,2011,Carotenoids and their isomers:color pigments in fruits and vegetables,Molecules,16(2):1710-1738
Liang J.Y.,An X.N.,Jiang J.X.,Zhu L.W.,and Zhang W.M.,2003,Study on the chemical constituents of the pod of Gleditsia sinensis,Zhongguo Yesheng Zhiwu Ziyuan(Chinese Wild Plant Resources),22(3):44-46(梁静谊,安鑫南,蒋建新,朱莉伟,张卫明,2003,皂荚化学组成的研究,中国野生植物资源,22(3):44-46)
Liu X.H.,Sun H.Y.,Chen W.W.,Guo Q.G.,Li X.L.,and Liang G.L.,2016,Advances in studies on carotenoids in loquat fruit,Guoshu Xuebao(Journal of Fruit Science),33(7):874-881(刘小洪,孙海艳,陈薇薇,郭启高,李晓林,梁国鲁,2016,枇杷果实类胡萝卜素研究进展,果树学报,33(7):874-881)
Mortazavi A.,Williams B.A.,McCue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nat.Methods,5(7):621-628
Sandmann G.,2015,Carotenoids of biotechnological importance,Adv.Biochem.Eng.Biotechnol.,148:449-467
Yin T.M.,2010,A review of the genomic and gene cloning studies in trees,Yichuan(Hereditas),32(7):677-684(尹佟明,2010,林木基因组及功能基因克隆研究概述,遗传,32(7):677-684)
Yue J.,Lu X.,Zhang H.,Ge J.,Gao X.,and Liu Y.,2017,Identification of conserved and novel microRNAs in blueberry,Front.Plant Sci.,8:1155
Zhang J.P.,Tian X.H.,Yang Y.X.,Liu Q.X.,Wang Q.,Chen L.P.,Li H.L.,and Zhang W.D.,2016,Gleditsia species:an ethnomedical,phytochemical and pharmacological review,J.Ethnopharmacol.,178:155-171
Zhang X.,Zheng Y.,Jagadeeswaran G.,Ren R.,Sunkar R.,and Jiang H.,2014,Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes,Insect Biochem.Mol.Biol.,47:12-22
Zhu J.,Chai L.,Hou B.Z.,Shen Y.Y.,and Guo J.X.,2015,Research advances and prospects of ABA in fleshy fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),42(9):1664-1672(祝军,柴璐,侯柄竹,沈元月,郭家选,2015,果实中脱落酸的研究进展与展望,园艺学报,42(9):1664-1672)