基于转录组学的不同色系蝴蝶兰花色苷差异积累分析
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摘要
蝴蝶兰花色丰富且极具变化,是研究花着色机制的理想材料。本研究利用RNA Sequencing(RNA-Seq)技术比较三种不同色系蝴蝶兰花器官转录组,鉴定蝴蝶兰花色苷合成的相关基因,从转录组水平揭示蝴蝶兰花色苷生物合成的分子调控机制。选取白花蝴蝶兰(PAPW)、红花蝴蝶兰(PAPR)和黄花蝴蝶兰(PAPY)为试验材料,对其花苞及盛开花朵分别釆样,分别提取三个样品的总RNA,利用Illumina Hiseq 2 000进行测序分析,过滤处理后分别得到总Clean reads片段为60 031 912、57 685 822和55 765 402个,GC含量分别为47.78%、47.36%和49.48%,平均长度约为575 bp,N50长度为940 bp,对获得的All-unigenes进行功能注释,注释到Swiss-Prot、Nt、KO、Nr、GO和KOG数据库的Unigenes分别是22 321、19 199、8 671、28 952、20 152和10 380个;PAPR和PAPW相比差异基因共有642个,上调基因有354个,下调基因有288个;PAPY和PAPW相比差异基因共有2 459个,上调基因有132个,下调基因2 327个;KEGG代谢分析表明,2个对比组中的差异基因富集在不同代谢通路中。PAPR和PAPW差异基因主要富集在类黄酮生物合成,苯丙氨酸代谢和钙信号通路等代谢通路,其中类黄酮生物合成中差异基因为8个(7个上调,1个下调)。PAPY和PAPW差异表达基因主要富集在DNA复制,烷、哌啶和吡啶生物碱和细胞凋亡等代谢通路,其中,参与到淀粉与蔗糖代谢中的差异基因最多,有34条(1个上调,33个下调)。这些信息为蝴蝶兰不同花色苷形成相关关键基因的研究提供了重要依据。
Phalaenopsis is rich in col or and varies greatly,which is an ideal material for studying the coloring mechanism of flowers.In this study,we used RNA sequencing(RNA-Seq) technology to compare the flower organs of three different-colored phalaenopsis,identify the genes related to anthocyanin biosynthesis in phalaenopsis and reveal the molecular regulation mechanism of anthocyanin biosynthesis in phalaenopsis from transcriptome level.White flower phalaenopsis(PAPW),red flower phalaenopsis(PAPR) and yellow flower phalaenopsis(PAPY) were selected as test materials,and their buds and flowers were sampled.The total RNA of three samples were extractedand Illumina Hiseq 2 000 was used to sequence.After filtration treatment,the total Clean reads fragments were 60031 912,57 685 822 and 55 765 402,respectively.The GC contents were 47.78%,47.36% and 49.48%,respectively,with the average length of 575 bp and N50 length of 940 bp.Functional annotation was carried out on the acquired All-unigenes,the results of which showed that the number of Unigenes of Swiss-Prot,Nt,KO,Nr,GO and KOG database were 22 321,19 199,8 671,28 952,20 152 and 10 380,respectively.A total of 642 differentially expressed genes(DEGs) were screened out from PAPR vs PAPW,in which 354 DEGs were up-regulated and 288 DEGs were down-regulated.A total of 2 459 DEGs were screened out from PAPY vs PAPW,including132 up-regulated and 2 327 down-regulated.The KEGG metabolism analysis indicated that these DEGs from two contrast groups gathered in different metabolic pathways.The DEGs from PAPR vs PAPW mainly gathered in metabolic pathways such as flavonoid biosynthesis,phenylalanine metabolism and calcium signaling pathways,and there were 8 DEGs in flavonoid biosynthesis pathways(7 up-regulated and 1 down-regulated genes).While the DEGs from PAPY vs PAPW mostly gathered in metabolic pathways such as DNA replication,alkane,piperidine and pyridine alkaloids and cell apoptosis.The DEGs involved in starch and sucrose metabolism were the most,with the number of 34(1 up-regulated gene and 33 down-regulated genes).These information could provide important reference for the study of key genes related to anthocyanin formation in phalaenopsis.
Phalaenopsis is rich in col or and varies greatly,which is an ideal material for studying the coloring mechanism of flowers.In this study,we used RNA sequencing(RNA-Seq) technology to compare the flower organs of three different-colored phalaenopsis,identify the genes related to anthocyanin biosynthesis in phalaenopsis and reveal the molecular regulation mechanism of anthocyanin biosynthesis in phalaenopsis from transcriptome level.White flower phalaenopsis(PAPW),red flower phalaenopsis(PAPR) and yellow flower phalaenopsis(PAPY) were selected as test materials,and their buds and flowers were sampled.The total RNA of three samples were extractedand Illumina Hiseq 2 000 was used to sequence.After filtration treatment,the total Clean reads fragments were 60031 912,57 685 822 and 55 765 402,respectively.The GC contents were 47.78%,47.36% and 49.48%,respectively,with the average length of 575 bp and N50 length of 940 bp.Functional annotation was carried out on the acquired All-unigenes,the results of which showed that the number of Unigenes of Swiss-Prot,Nt,KO,Nr,GO and KOG database were 22 321,19 199,8 671,28 952,20 152 and 10 380,respectively.A total of 642 differentially expressed genes(DEGs) were screened out from PAPR vs PAPW,in which 354 DEGs were up-regulated and 288 DEGs were down-regulated.A total of 2 459 DEGs were screened out from PAPY vs PAPW,including132 up-regulated and 2 327 down-regulated.The KEGG metabolism analysis indicated that these DEGs from two contrast groups gathered in different metabolic pathways.The DEGs from PAPR vs PAPW mainly gathered in metabolic pathways such as flavonoid biosynthesis,phenylalanine metabolism and calcium signaling pathways,and there were 8 DEGs in flavonoid biosynthesis pathways(7 up-regulated and 1 down-regulated genes).While the DEGs from PAPY vs PAPW mostly gathered in metabolic pathways such as DNA replication,alkane,piperidine and pyridine alkaloids and cell apoptosis.The DEGs involved in starch and sucrose metabolism were the most,with the number of 34(1 up-regulated gene and 33 down-regulated genes).These information could provide important reference for the study of key genes related to anthocyanin formation in phalaenopsis.
引文
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Dai S.L.,and Homg Y.,2016,Molecular breeding for flower colors modification on ornamental plants based on the mechanism of anthocyanins biosynthesis and coloration,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(3):529-542(戴思兰,洪艳,2016,基于花青素苷合成和呈色机理的观赏植物花色改良分子育种,中国农业科学,49(3):529-542)
Ding L.,Li C.H.,Huang S.H.,and Zhang Z.Q.,2015,Research advances of flavonoid 3'-hydroxylase gene in ornamental plant,Beifang Yuanyi(Northern Horticulture),(17):188-193(丁灵,李崇晖,黄少华,张志群,2015,调控观赏植物花色的类黄酮3'羟化酶基因研究进展,北方园艺,(17):188-193)
Feng H.,Yi S.L.,Zuo J.Q.,Xie J.H.,and Huang X.,2014Progress 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)
Gandíaherrero F.,and Garcíacarmona F.,2013,Biosynthesis of betalains:yellow and violet plant pigments,Trends in Plant Science,18(6):1-10
Han K.T.,Hu K.,and Dai S.L.,2008,Flower color breeding by molecular design in ornamentals,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(1):16-24(韩科厅,胡可,戴思兰,2008,观赏植物花色的分子设计,分子植物育种,6(1):16-24)
Han Y.,Vimolmangkang S.,Soria-Guerra R.E.,Rosales-Mendoza S.,Zheng D.,Lygin A.V.,and Korban S.S.,2010,Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress,Plant Physiol.,153(2):806-820
Hou J.,Tong L.,Cui G.X.,Xu Z.R.,and Li Y.H.,2011,Research advances of plant flavonoid 3'-hydroxylase(F3'H)gene,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(7):641-647(侯杰,佟玲,崔国新,许志茹,李玉花,2011,植物类黄酮3'-羟化酶(F3'H)基因的研究进展,植物生理学报,47(7):641-647)
Huang C.G.,and Ma S.X.,2012,The over-expression of dihydroflavonol-4-reductase(DFR)in tobacco,Shanxi Nongye Kexue(Journal of Shanxi Agricultural Sciences),40(6):563-565(黄春国,马素娴,2012,二氢黄酮醇-4-还原酶(DFR)基因在烟草中的过量表达,山西农业科学,40(6):563-565)
Huang J.Z.,Lin C.P.,Cheng T.C.,Chang C.H.,Cheng S.Y.Chen Y.W.,and Chen F.C.,2015,A de novo floral transcriptome reveals clues into phalaenopsis orchid flower development,PLo S One,10(5):e0123474
Jing T.,Wang L.,Tana W.N.,and Du H.Y.,2016,Analysis of digital gene expression about eucommia ulmoides oliver leaves at different developmental stages,Xibei Zhiwu Xubao(Acta Botanica Boreali-Occidentalia Sinica),36(2):257-265(荆腾王淋,乌云塔娜,杜红岩,2016,杜仲叶片不同发育时期数字基因表达谱分析,西北植物学报,36(2):257-265)
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