基于转录组测序的云南‘火把梨’花青素相关基因分析
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摘要
本研究以云南‘火把梨’转色前后的梨果皮为试验材料,采用高通量测序技术(Illumina HiSeq TM 2000/MiSeq)对两种梨材料全转录功能基因组测序后组装,再通过GO、Swiss-Prot两大数据库进行花青素相关差异表达基因筛选,以了解可能与花青素合成相关的基因及其表达模式,同时将差异基因在NCBI数据库进行Blast序列比对,对其功能进行分析。最终共获得7.52 Gb数据,组装过滤后共获得75 227 538条Clean reads,3 920条差异表达基因,通过通路分析和Pathway功能注释,在花青素合成过程中的两条通路中共注释到30条与花青素合成相关的差异表达基因,初步对这些差异基因的功能进行鉴定和分析,对后期基因克隆及转基因等研究具有重要意义。
In this research, the peels of Yunnan 'Huoba Pear' before and after color transformation was used as experimental materials, the full transcription functional genomes of the two pear materials were sequenced and assembled using high-throughput sequencing technology(Illumina HiSeqTM2000/MiSeq), and then anthocyaninrelated differentially expressed genes were screened through GO and Swiss-Prot databases to understand the genes and their expression patterns that might be associated with anthocyanin synthesis. At the same time, the Blast sequences of differential genes were compared in the NCBI database and their functions were analyzed. A total of7.52 Gb data was finally obtained. After assembly and filtration, 75 227 538 Clean reads and 3 920 differentially expressed genes were obtained. Through pathway analysis and Pathway function annotation, 30 differentially expressed genes related to anthocyanin synthesis were annotated in the two pathways during anthocyanin synthesis. The preliminary identification and analysis of the functions of these differential genes might be of great significance to the further gene cloning and transgenic resrarch work.
In this research, the peels of Yunnan 'Huoba Pear' before and after color transformation was used as experimental materials, the full transcription functional genomes of the two pear materials were sequenced and assembled using high-throughput sequencing technology(Illumina HiSeqTM2000/MiSeq), and then anthocyaninrelated differentially expressed genes were screened through GO and Swiss-Prot databases to understand the genes and their expression patterns that might be associated with anthocyanin synthesis. At the same time, the Blast sequences of differential genes were compared in the NCBI database and their functions were analyzed. A total of7.52 Gb data was finally obtained. After assembly and filtration, 75 227 538 Clean reads and 3 920 differentially expressed genes were obtained. Through pathway analysis and Pathway function annotation, 30 differentially expressed genes related to anthocyanin synthesis were annotated in the two pathways during anthocyanin synthesis. The preliminary identification and analysis of the functions of these differential genes might be of great significance to the further gene cloning and transgenic resrarch work.
引文
Ageorges A.,Fernandez L.,Vialet S.,Merdinoglu D.,Terrier N.,and Romieu C.,2006,Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries,Plant Sci.,170(2):372-383
Allan A.C.,Hellens R.P.,and Laing W.A.,2008,MYB transcription factors that colour our fruit,Tren ds Plant Sci.,13(3):99-102
Anders S.,and Huber W.,2010,Differential expression analysis for sequence count data,Genome Biol.,11(10):R106
Dussi M.C.,Sugar D.,and Wrolstad R.E.,1995,Characterizing and quantifying anthocyanins in red pears and the effect of light quality on fruit color,American Society for Horticultural Science,120(5):785-789
Feng S.Q.,Chen X.S.,Zhang C.Y.,Liu X.J.,Liu Z.C.,Wang H.B.,Wang Y.L.,and Zhou C.H.,2008,A study of the relationship between anthocyanin biosynthesis and related enzymes activity in Pyrus pyrifolia'mantianhong'and its bud sports'aoguan',Zhongguo Nongye Kexue(Scientia Agricultura Sinica),41(10):3184-3190(冯守千,陈学森,张春雨,刘晓静,刘遵春,王海波,王延玲,周朝华,2008,砂梨品种‘满天红’及其芽变品系‘奥冠’花青苷合成与相关酶活性研究,中国农业科学,41(10):3184-3190)
Feng S.Q.,Wang Y.L.,Yang S.,Xu Y.T.,and Chen X.S.,2010,Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10,Planta,232(1):245-255
Fischer T.C.,Gosch C.,Pfeiffer J.,Halbwirth H.,Halle C.,Stich K.,and Forkmann G.,2007,Flavonoid genes of pear(Pyrus communis),Trees,21(5):521-529
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.D.,Chen Z.H.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma F.D.,Birren B.,Nusbaum C.,Toh K.L.,Friedman N.,and Regev A.,2011,Full-length transcriptome assembly from RNA-Seq data without a reference genome,Nat.Biotech.,29(7):644-652
Huang C.H.,Yu B.,Su J.,Shu Q.,and Teng Y.W.,2010,A study on coloration physiology of fruit in two red chinese sand pear cultivars'Meirensu'and'Yunhongli No.1',Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(7):1433-1440(黄春辉,俞波,苏俊,舒群,滕元文,2010,‘美人酥’和‘云红梨1号’红皮砂梨果实的着色生理,中国农业科学,43(7):1433-1440)
Jia C.L.,Zhang Y.,Zhu L.,and Zhang Y.,2015,Application progress of transcriptome sequencing technology in biological sequencing,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(10):2388-2394(贾昌路,张瑶,朱玲,张悦,2015,转录组测序技术在生物测序中的应用研究进展,分子植物育种,13(10):2388-2394)
Petroni K.,and Tonelli C.,2011,Recent advances on the regulation of anthocyanin synthesis in reproductive organs,Plant Biol.,181(3):219-229
Pierantoni L.,Dondini L.,Franceschi P.D.,Musacchi S.,Winkel B.S.J.,and Sansavini S.,2010,Mapping of an anthocyaninregulating MYB transcription factor and its expression in red and green pear,Pyrus communis,Plant Physiol.Biochem.,48(12):1020-1026
Sangwan R.S.,Tripathi S.,Singh J.,Narnoliya L.K.,and Sangwan N.S.,2013,De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural,functional and regulatory genes with special reference to secondary metabolism,Gene,525(1):58-76
Steyn W.J.,Holcroft D.M.,Wand S.J.E.,and Jacobs G.,2004a,Anthocyanin degradation in detached pome fruit with reference to preharvest red color loss and pigmentation patterns of blushed and fully red pears,Journal of the American Society for Horticultural Science,129(1):13-19
Steyn W.J.,Holcroft D.M.,Wand S.J.E.,and Jacobs G.,2004b,Regulation of pear color development in relation to activity of flavonoid enzymes,Journal of the American Society for Horticultural Science,129(1):6-12
Sun X.,Han J.,Fang J.G.,Shangguan L.F.,Wang X.C.,Song C.N.,and Li X.Y.,2012,Important research progress of coloring molecular mechanisms in grape berry,Zhiwu Shengli Xuebao(Physiology Journal Plant Physiology Journal),48(4):333-342(孙欣,韩键,房经贵,上官凌飞,王西成,宋长年,李晓颖,2012,葡萄浆果着色分子机理的重要研究进展,植物生理学报,48(4):333-342)
Tian P.,Su Y.L.,Kang B.S.,and Wei W.D.,2015,Analyses of expression patterns of transcription factor MYB10 and anthocyanin synthesis genes in two red skin pear varieties,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Science),31(1):166-171(田鹏,苏艳丽,康保珊,魏闻东,2015,两个红梨品种花色苷合成相关基因及转录因子MYB10表达模式分析,江苏农业学报,31(1):166-171)
Xiao C.C.,Li J.M.,Yao G.F.,Liu J.,Hu H.J.,Cao Y.F.,Zhang S.L.,and Wu J.,2014,Characteristics of components and contents of anthocyanin in peel of red-skinned pear fruits from different species,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),37(4):60-66(肖长城,李甲明,姚改芳,刘军,胡红菊,曹玉芬,张绍铃,吴俊,2014,不同红梨品种果皮中花色素苷组分及含量特征分析,南京农业大学学报,37(4):60-66)
Zabetakis I.,Leclerc D.,and Kajda P.,2000,The effect of high hydrostatic pressure on the strawberry anthocyanins,J.A-gric.Food Chem.,48(7):2749-2754
Zhang D.,and Teng Y.W.,2011,Germplasm resources of red pears and research progress in pear coloring mechanism,Guoshu Xuebao(Journal of Fruit Science),28(3):485-492(张东,滕元文,2011,红梨资源及其果实着色机制研究进展,果树学报,28(3):485-492)
Zhang Q.,Wang H.Q.,Leng P.,and Jia L.X.,2008,Mechanism of anthocyanins and flavonols in fruit development of strawberries,Yuanyi Xuebao(Acta Horticulturae Sinica),35(12):1735-1741(张琼,王红清,冷平,贾乐新,2008,草莓果实发育过程中花色苷和黄酮醇类物质的形成机制,园艺学报,35(12):1735-1741)
Zhang X.D.,Allan A.C.,Yi Q.,Chen L.M.,Li K.Z.,Shu Q.,and Su J.,2011,Differential Gene expression analysis of Yunnan red pear,Pyrus pyrifolia,during fruit skin coloration,Plant Mol.Biol.Rep.,29(2):305-314
Zhang X.D.,Chen L.M.,Shu Q.,Su J.,and Li K.Z.,2009,Effect of light on the peel coloration and fruit quality of Yunnan red pear,Zhongguo Nanfang Guoshu(South China Fruits),38(5):1-6(张晓东,陈丽梅,舒群,苏俊,李昆志,2009,光照对云南红梨着色及果实品质影响的研究,中国南方果树,38(5):1-6)
Zhou J.,Xin P.Y.,Xu Y.L.,Tao P.,and Shu Q.,2010,Current advance of red skin pear resource origin from China and molecular mechanism of red development in pear skint,Beifang Yuanyi(Northern Horticulture),(6):213-218(周军,辛培尧,许玉兰,陶磅,舒群,2010,红皮梨资源及红色发育分子机制的研究,北方园艺,(6):213-218)
Zhu T.T.,Liang D.,and Xia H.,2016,Progress in the study of the regulation of anthocyanin synthesis by R2R3-MYB in fruits,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):985-991(朱婷婷,梁东,夏惠,2016,R2R3-MYB调控果实花色苷合成的研究进展,基因组学与应用生物学,35(4):985-991)
Allan A.C.,Hellens R.P.,and Laing W.A.,2008,MYB transcription factors that colour our fruit,Tren ds Plant Sci.,13(3):99-102
Anders S.,and Huber W.,2010,Differential expression analysis for sequence count data,Genome Biol.,11(10):R106
Dussi M.C.,Sugar D.,and Wrolstad R.E.,1995,Characterizing and quantifying anthocyanins in red pears and the effect of light quality on fruit color,American Society for Horticultural Science,120(5):785-789
Feng S.Q.,Chen X.S.,Zhang C.Y.,Liu X.J.,Liu Z.C.,Wang H.B.,Wang Y.L.,and Zhou C.H.,2008,A study of the relationship between anthocyanin biosynthesis and related enzymes activity in Pyrus pyrifolia'mantianhong'and its bud sports'aoguan',Zhongguo Nongye Kexue(Scientia Agricultura Sinica),41(10):3184-3190(冯守千,陈学森,张春雨,刘晓静,刘遵春,王海波,王延玲,周朝华,2008,砂梨品种‘满天红’及其芽变品系‘奥冠’花青苷合成与相关酶活性研究,中国农业科学,41(10):3184-3190)
Feng S.Q.,Wang Y.L.,Yang S.,Xu Y.T.,and Chen X.S.,2010,Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10,Planta,232(1):245-255
Fischer T.C.,Gosch C.,Pfeiffer J.,Halbwirth H.,Halle C.,Stich K.,and Forkmann G.,2007,Flavonoid genes of pear(Pyrus communis),Trees,21(5):521-529
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.D.,Chen Z.H.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma F.D.,Birren B.,Nusbaum C.,Toh K.L.,Friedman N.,and Regev A.,2011,Full-length transcriptome assembly from RNA-Seq data without a reference genome,Nat.Biotech.,29(7):644-652
Huang C.H.,Yu B.,Su J.,Shu Q.,and Teng Y.W.,2010,A study on coloration physiology of fruit in two red chinese sand pear cultivars'Meirensu'and'Yunhongli No.1',Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(7):1433-1440(黄春辉,俞波,苏俊,舒群,滕元文,2010,‘美人酥’和‘云红梨1号’红皮砂梨果实的着色生理,中国农业科学,43(7):1433-1440)
Jia C.L.,Zhang Y.,Zhu L.,and Zhang Y.,2015,Application progress of transcriptome sequencing technology in biological sequencing,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(10):2388-2394(贾昌路,张瑶,朱玲,张悦,2015,转录组测序技术在生物测序中的应用研究进展,分子植物育种,13(10):2388-2394)
Petroni K.,and Tonelli C.,2011,Recent advances on the regulation of anthocyanin synthesis in reproductive organs,Plant Biol.,181(3):219-229
Pierantoni L.,Dondini L.,Franceschi P.D.,Musacchi S.,Winkel B.S.J.,and Sansavini S.,2010,Mapping of an anthocyaninregulating MYB transcription factor and its expression in red and green pear,Pyrus communis,Plant Physiol.Biochem.,48(12):1020-1026
Sangwan R.S.,Tripathi S.,Singh J.,Narnoliya L.K.,and Sangwan N.S.,2013,De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural,functional and regulatory genes with special reference to secondary metabolism,Gene,525(1):58-76
Steyn W.J.,Holcroft D.M.,Wand S.J.E.,and Jacobs G.,2004a,Anthocyanin degradation in detached pome fruit with reference to preharvest red color loss and pigmentation patterns of blushed and fully red pears,Journal of the American Society for Horticultural Science,129(1):13-19
Steyn W.J.,Holcroft D.M.,Wand S.J.E.,and Jacobs G.,2004b,Regulation of pear color development in relation to activity of flavonoid enzymes,Journal of the American Society for Horticultural Science,129(1):6-12
Sun X.,Han J.,Fang J.G.,Shangguan L.F.,Wang X.C.,Song C.N.,and Li X.Y.,2012,Important research progress of coloring molecular mechanisms in grape berry,Zhiwu Shengli Xuebao(Physiology Journal Plant Physiology Journal),48(4):333-342(孙欣,韩键,房经贵,上官凌飞,王西成,宋长年,李晓颖,2012,葡萄浆果着色分子机理的重要研究进展,植物生理学报,48(4):333-342)
Tian P.,Su Y.L.,Kang B.S.,and Wei W.D.,2015,Analyses of expression patterns of transcription factor MYB10 and anthocyanin synthesis genes in two red skin pear varieties,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Science),31(1):166-171(田鹏,苏艳丽,康保珊,魏闻东,2015,两个红梨品种花色苷合成相关基因及转录因子MYB10表达模式分析,江苏农业学报,31(1):166-171)
Xiao C.C.,Li J.M.,Yao G.F.,Liu J.,Hu H.J.,Cao Y.F.,Zhang S.L.,and Wu J.,2014,Characteristics of components and contents of anthocyanin in peel of red-skinned pear fruits from different species,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),37(4):60-66(肖长城,李甲明,姚改芳,刘军,胡红菊,曹玉芬,张绍铃,吴俊,2014,不同红梨品种果皮中花色素苷组分及含量特征分析,南京农业大学学报,37(4):60-66)
Zabetakis I.,Leclerc D.,and Kajda P.,2000,The effect of high hydrostatic pressure on the strawberry anthocyanins,J.A-gric.Food Chem.,48(7):2749-2754
Zhang D.,and Teng Y.W.,2011,Germplasm resources of red pears and research progress in pear coloring mechanism,Guoshu Xuebao(Journal of Fruit Science),28(3):485-492(张东,滕元文,2011,红梨资源及其果实着色机制研究进展,果树学报,28(3):485-492)
Zhang Q.,Wang H.Q.,Leng P.,and Jia L.X.,2008,Mechanism of anthocyanins and flavonols in fruit development of strawberries,Yuanyi Xuebao(Acta Horticulturae Sinica),35(12):1735-1741(张琼,王红清,冷平,贾乐新,2008,草莓果实发育过程中花色苷和黄酮醇类物质的形成机制,园艺学报,35(12):1735-1741)
Zhang X.D.,Allan A.C.,Yi Q.,Chen L.M.,Li K.Z.,Shu Q.,and Su J.,2011,Differential Gene expression analysis of Yunnan red pear,Pyrus pyrifolia,during fruit skin coloration,Plant Mol.Biol.Rep.,29(2):305-314
Zhang X.D.,Chen L.M.,Shu Q.,Su J.,and Li K.Z.,2009,Effect of light on the peel coloration and fruit quality of Yunnan red pear,Zhongguo Nanfang Guoshu(South China Fruits),38(5):1-6(张晓东,陈丽梅,舒群,苏俊,李昆志,2009,光照对云南红梨着色及果实品质影响的研究,中国南方果树,38(5):1-6)
Zhou J.,Xin P.Y.,Xu Y.L.,Tao P.,and Shu Q.,2010,Current advance of red skin pear resource origin from China and molecular mechanism of red development in pear skint,Beifang Yuanyi(Northern Horticulture),(6):213-218(周军,辛培尧,许玉兰,陶磅,舒群,2010,红皮梨资源及红色发育分子机制的研究,北方园艺,(6):213-218)
Zhu T.T.,Liang D.,and Xia H.,2016,Progress in the study of the regulation of anthocyanin synthesis by R2R3-MYB in fruits,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(4):985-991(朱婷婷,梁东,夏惠,2016,R2R3-MYB调控果实花色苷合成的研究进展,基因组学与应用生物学,35(4):985-991)
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