红肉猕猴桃果实发育期不同果肉部位比较转录组分析
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摘要
红肉猕猴桃中果皮和内果皮在果实发育不同时期色泽变化模式有显著差异,但目前对于其内在的分子调控机制尚不清楚。本研究中,利用高通量测序技术,对红肉猕猴桃(‘红实2号’)中果皮与内果皮4个不同发育时期(开花后0 d, 28 d, 75 d, 157 d,分别标注为T1时期, T2时期, T3时期, T4时期)的样本进行转录组测序。经去除低质量数据后,得到Clean reads数目为2 192～4 331万,检测到的基因数目为24 607～29 170个。在(log2 Ratio)≥1和FDR<0.05阈值下,中果皮和内果皮在T2、T3、T4时期检测到的差异基因数目分别为1 880个、4 799个、7 346个和4 31个、1 493个、7 700个。KEGG分析显示这些差异表达基因被富集到68个代谢途径,包括DNA复制、RNA转运、蛋白质输出、植物激素信号转导、类黄酮生物合成、花青素合成、淀粉与蔗糖的代谢等。此外我们还鉴定到13个花青素合成相关基因差异表达,大部分基因的表达量随果实的发育呈现下降趋势,这一结果与花青素含量变化模式相吻合。本研究不仅深化了对猕猴桃果实成色差异机理的认识,还对今后的彩色猕猴桃选育种工作有一定的帮助。
There are big differences in color change patterns of red-fleshed kiwifruit sarcocarp and endocarp during different fruit development stages, but the innate molecular mechanism is poorly known. In the present study, using high-throughput sequencing technology, we systematically sequenced four stages(0 day, 28 days, 75 days and 157 days after flowering, termed as T1 stage, T2 stage, T3 stage, T4 stage) of red-fleshed kiwifruit('Hongshi 2')sarcocarp and endocarp. After removing low quality data, a total of 21.92～43.31 million clean reads were generated, which detected 24 607～29 170 kiwifruit genes. Under the limited role((log2 Ratio) ≥1 and FDR<0.05),1 880, 4 799, 7 346 and 431, 1 493, 7 700 differentially expressed genes were detected in T2, T3, T4 stages of sarcocarp and endocarp. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis showed 68 biological pathways were significantly enriched, including DNA replication, RNA transport, protein export, plant hormone signal transduction, flavonoid biosynthesis, anthocyanin biosynthesis, starch and sucrose metabolism, etc. 13 differentially expressed genes related to anthocyanin biosynthesis were further detected. Most of them expression patterns were decrease during fruit development, which was consistent with the change of anthocyanin content.The research present here not only broadens the mechanism of color formation in kiwifruit fruits, but also contributes to the color kiwifruit molecular breeding in future.
There are big differences in color change patterns of red-fleshed kiwifruit sarcocarp and endocarp during different fruit development stages, but the innate molecular mechanism is poorly known. In the present study, using high-throughput sequencing technology, we systematically sequenced four stages(0 day, 28 days, 75 days and 157 days after flowering, termed as T1 stage, T2 stage, T3 stage, T4 stage) of red-fleshed kiwifruit('Hongshi 2')sarcocarp and endocarp. After removing low quality data, a total of 21.92～43.31 million clean reads were generated, which detected 24 607～29 170 kiwifruit genes. Under the limited role((log2 Ratio) ≥1 and FDR<0.05),1 880, 4 799, 7 346 and 431, 1 493, 7 700 differentially expressed genes were detected in T2, T3, T4 stages of sarcocarp and endocarp. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis showed 68 biological pathways were significantly enriched, including DNA replication, RNA transport, protein export, plant hormone signal transduction, flavonoid biosynthesis, anthocyanin biosynthesis, starch and sucrose metabolism, etc. 13 differentially expressed genes related to anthocyanin biosynthesis were further detected. Most of them expression patterns were decrease during fruit development, which was consistent with the change of anthocyanin content.The research present here not only broadens the mechanism of color formation in kiwifruit fruits, but also contributes to the color kiwifruit molecular breeding in future.
引文
Chen D.,Song H.Y.,Gong R.G.,Li J.,Tu M.Y.,Jiang G.L.,Sun S.X.,and Tang H.R.,2017,Comparative transcriptome analysis of different stages of fruit ripening between red-and white-flesh loquat,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2112-2118(陈栋,宋海岩,龚荣高,李靖涂美艳,江国良,孙淑霞,汤浩茹,2017,红肉枇杷和白肉枇杷果实成熟不同阶段的比较转录组研究,分子植物育种,15(6):2112-2118)
Friso G.,and van Wijk K.J.,2015,Posttranslational protein modifications in plant metabolism,Plant Physiol.,169(3):1469-1487
Huang S.X.,Ding J.,Deng D.,Tang W.,Sun H.H.,Liu D.Y.Zhang L.,Niu X.L.,Zhang X.,Meng M.,Yu J.D.,Liu J.Han Y.,Shi W.,Zhang D.F.,Cao S.Q.,Wei Z.J.,Cui Y.L.Xia Y.H.,Zeng H.P.,Bao K.,Lin L.,Min Y.,Zhang H.Miao M.,Tang X.F.,Zhu Y.,Sui Y.Y.,Li G.W.,Sun H.J.Yue J.Y.,Sun J.Q.,Liu F.F.,Zhou L.Q.,Lei L.,Zheng X.Q.Liu M.,Huang L.,Song J.,Xu C.H.,Li J.W.,Ye K.Y.Zhong S.L.,Lu B.R.,He G.H.,Xiao F.M.,Wang H.L.Zheng H.K.,Fei Z.J.,and Liu Y.S.,2013,Draft genome of the kiwifruit Actinidia chinensis,Nat.Commun.,4(4):2640
Labaj P.P.,and Kreil D.P.,2016,Sensitivity,specificity,and reproducibility of RNA-Seq differential expression calls,Biol.Direct,11(1):66
Li S.N.,Wang W.Y.,Gao J.L.,Yin K.Q.,Wang R.,Wang C.C.,Petersen M.,Mundy J.,and Qiu J.L.,2016,MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis,Plant Cell,28(11):2866
Li W.B.,Liu Y.F.,Zeng S.H.,Xiao G.,Wang G.,Wang Y.,Peng M.,and Huang H.W.,2015,Gene expression profiling of development and anthocyanin accumulation in kiwifruit(Actinidia chinensis)based on transcriptome sequencing,PLoS One,10(8):e0136439
Li Y.K.,Fang J.B.,Qi X.J.,Lin M.M.,Zhong Y.P.,and Sun L.M.,2018a,A key structural gene,AaLDOX,is involved in anthocyanin biosynthesis in all red-fleshed kiwifruit(Actinidia arguta)based on transcriptome analysis,Gene,648:31-41
Li Y.K.,Fang J.B.,Qi X.J.,Lin M.M.,Zhong Y.P.,Sun L.M.,and Cui W.,2018b,Combined analysis of the fruit metabolome and transcriptome reveals candidate genes involved in flavonoid biosynthesis in Actinidia arguta,Int.J.Mol.Sci.,19(5):1471
Man Y.P.,2012,Cloning and expression analysis of genes that were responsible for anthocyanin biosynthesis and transportation in red-fleshed kiwifruit,Thesis for M.S.,SouthCentral University for Nationalities,Supervisor:Qin R.,pp.13-28(满玉萍,2012,红肉猕猴桃花青素合成及运转相关基因的克隆及表达分析,硕士学位论文,中南民族大学,导师:覃瑞,pp.13-28)
Montefiori M.,McGhie T.K.,Costa G.,and Ferguson A.R.,2005,Pigments in the fruit of red-fleshed kiwifruit(Actinidia chinensis and Actinidia deliciosa),J.Agric.Food Chem.,53(24):9526-9530
Pan D.L.,Zhang J.Y.,Wang T.,Wang G.,Jia Z.H.,and Guo Z.R.,2017,Morphological diversity comparison of female flowers among 18 cultivars of kiwifruit,Tianjin Nongye Kexue(Tianjin Agricultural Sciences),23(12):85-87(潘德林,张计育,王涛,王刚,贾展慧,郭忠仁,2017,18个猕猴桃优良品种雌花形态多样性比较,天津农业科学,23(12):85-87)
Petroni K.,Pilu R.,and Tonelli C.,2014,Anthocyanins in corn:a wealth of genes for human health,Planta,240(5):901-911
Petrussa E.,Braidot E.,Zancani M.,Peresson C.,Bertolini A.,Patui S.,and Vianello A.,2013,Plant flavonoids-biosynthesis,transport and involvement in stress responses,Int.J.Mol.Sci.,14(7):14950-14973
Qi X.J.,Xu S.K.,Lin M.M.,Li Y.K.,Sun L.M.,and Fang J.B.,2015,Research advances on fruit coloring mechanism in red-fleshed kiwifruit,Guoshu Xuebao(Journal of Fruit Science),32(6):1232-1240(齐秀娟,徐善坤,林苗苗,李玉阔,孙雷明,方金豹,2015,红肉猕猴桃果实着色机制研究进展,果树学报,32(6):1232-1240)
Saito K.,Yonekura-Sakakibara K.,Nakabayashi R.,Higashi Y.Yamazaki M.,Tohge T.,and Fernie A.R.,2013,The flavonoid biosynthetic pathway in Arabidopsis:structural and genetic diversity,Plant Physiol.Biochem.,72:21-34
Vodopivec B.M.,Wang J.,M?ller A.L.,Krake J.,Lund T.Hansen P.E.,and Nielsen S.L.,2013,Differences in the structure of anthocyanins from the two amphibious plants Lobelia cardinalis and Nesaea crassicaulis,Nat.Prod.Res.27(7):654-664
Wang Z.,Gerstein M.,and Snyder M.,2009,RNA-Seq:a revolutionary tool for transcriptomics,Nat.Rev.Genet.,10(1):57-63
Xie Y.,Xia H.,Liang D.,Wang Y.Z.,Liu J.,Zhuang Q.G.,and Li M.Z.,2018,Genetic diversity analysis of 25 Actinidia materials and the establishment of DNA fingerprint,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(15):5001-5007(谢玥,夏惠,梁东,王永志,刘娟,庄启国,李明章,2018,25个猕猴桃材料遗传多样性分析及DNA指纹图谱的建立,分子植物育种,16(15):5001-5007)
Yang J.,Jiang Z.W.,and Wang Y.C.,2010,Cloning and expression of dihydroflavonol 4-reductase in Actinidia chinensis var.rufopulpa,Wuhan Zhiwuxue Yanjiu(Journal of Wuhan Botanical Research),28(6):673-681(杨俊,姜正旺,王彦昌,2010,红肉猕猴桃DFR基因的克隆及表达分析,武汉植物学研究,28(6):673-681)
Zhang L.,Man Y.P.,Jiang Z.W.,and Wang Y.C.,2012,Cloning and expression of anthocyanin pathway genes,AcCHS and AcLDOX,in Actinidia chinensis,Yuanyi Xuebao(Acta Horticulturae Sinica),39(11):2124-2132(张亮,满玉萍,姜正旺,王彦昌,2012,猕猴桃花青素合成途径基因AcCHS和AcLDOX的克隆与表达分析,园艺学报,39(11):2124-2132)
Zhao Y.C.,Liu X.H.,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)
Friso G.,and van Wijk K.J.,2015,Posttranslational protein modifications in plant metabolism,Plant Physiol.,169(3):1469-1487
Huang S.X.,Ding J.,Deng D.,Tang W.,Sun H.H.,Liu D.Y.Zhang L.,Niu X.L.,Zhang X.,Meng M.,Yu J.D.,Liu J.Han Y.,Shi W.,Zhang D.F.,Cao S.Q.,Wei Z.J.,Cui Y.L.Xia Y.H.,Zeng H.P.,Bao K.,Lin L.,Min Y.,Zhang H.Miao M.,Tang X.F.,Zhu Y.,Sui Y.Y.,Li G.W.,Sun H.J.Yue J.Y.,Sun J.Q.,Liu F.F.,Zhou L.Q.,Lei L.,Zheng X.Q.Liu M.,Huang L.,Song J.,Xu C.H.,Li J.W.,Ye K.Y.Zhong S.L.,Lu B.R.,He G.H.,Xiao F.M.,Wang H.L.Zheng H.K.,Fei Z.J.,and Liu Y.S.,2013,Draft genome of the kiwifruit Actinidia chinensis,Nat.Commun.,4(4):2640
Labaj P.P.,and Kreil D.P.,2016,Sensitivity,specificity,and reproducibility of RNA-Seq differential expression calls,Biol.Direct,11(1):66
Li S.N.,Wang W.Y.,Gao J.L.,Yin K.Q.,Wang R.,Wang C.C.,Petersen M.,Mundy J.,and Qiu J.L.,2016,MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis,Plant Cell,28(11):2866
Li W.B.,Liu Y.F.,Zeng S.H.,Xiao G.,Wang G.,Wang Y.,Peng M.,and Huang H.W.,2015,Gene expression profiling of development and anthocyanin accumulation in kiwifruit(Actinidia chinensis)based on transcriptome sequencing,PLoS One,10(8):e0136439
Li Y.K.,Fang J.B.,Qi X.J.,Lin M.M.,Zhong Y.P.,and Sun L.M.,2018a,A key structural gene,AaLDOX,is involved in anthocyanin biosynthesis in all red-fleshed kiwifruit(Actinidia arguta)based on transcriptome analysis,Gene,648:31-41
Li Y.K.,Fang J.B.,Qi X.J.,Lin M.M.,Zhong Y.P.,Sun L.M.,and Cui W.,2018b,Combined analysis of the fruit metabolome and transcriptome reveals candidate genes involved in flavonoid biosynthesis in Actinidia arguta,Int.J.Mol.Sci.,19(5):1471
Man Y.P.,2012,Cloning and expression analysis of genes that were responsible for anthocyanin biosynthesis and transportation in red-fleshed kiwifruit,Thesis for M.S.,SouthCentral University for Nationalities,Supervisor:Qin R.,pp.13-28(满玉萍,2012,红肉猕猴桃花青素合成及运转相关基因的克隆及表达分析,硕士学位论文,中南民族大学,导师:覃瑞,pp.13-28)
Montefiori M.,McGhie T.K.,Costa G.,and Ferguson A.R.,2005,Pigments in the fruit of red-fleshed kiwifruit(Actinidia chinensis and Actinidia deliciosa),J.Agric.Food Chem.,53(24):9526-9530
Pan D.L.,Zhang J.Y.,Wang T.,Wang G.,Jia Z.H.,and Guo Z.R.,2017,Morphological diversity comparison of female flowers among 18 cultivars of kiwifruit,Tianjin Nongye Kexue(Tianjin Agricultural Sciences),23(12):85-87(潘德林,张计育,王涛,王刚,贾展慧,郭忠仁,2017,18个猕猴桃优良品种雌花形态多样性比较,天津农业科学,23(12):85-87)
Petroni K.,Pilu R.,and Tonelli C.,2014,Anthocyanins in corn:a wealth of genes for human health,Planta,240(5):901-911
Petrussa E.,Braidot E.,Zancani M.,Peresson C.,Bertolini A.,Patui S.,and Vianello A.,2013,Plant flavonoids-biosynthesis,transport and involvement in stress responses,Int.J.Mol.Sci.,14(7):14950-14973
Qi X.J.,Xu S.K.,Lin M.M.,Li Y.K.,Sun L.M.,and Fang J.B.,2015,Research advances on fruit coloring mechanism in red-fleshed kiwifruit,Guoshu Xuebao(Journal of Fruit Science),32(6):1232-1240(齐秀娟,徐善坤,林苗苗,李玉阔,孙雷明,方金豹,2015,红肉猕猴桃果实着色机制研究进展,果树学报,32(6):1232-1240)
Saito K.,Yonekura-Sakakibara K.,Nakabayashi R.,Higashi Y.Yamazaki M.,Tohge T.,and Fernie A.R.,2013,The flavonoid biosynthetic pathway in Arabidopsis:structural and genetic diversity,Plant Physiol.Biochem.,72:21-34
Vodopivec B.M.,Wang J.,M?ller A.L.,Krake J.,Lund T.Hansen P.E.,and Nielsen S.L.,2013,Differences in the structure of anthocyanins from the two amphibious plants Lobelia cardinalis and Nesaea crassicaulis,Nat.Prod.Res.27(7):654-664
Wang Z.,Gerstein M.,and Snyder M.,2009,RNA-Seq:a revolutionary tool for transcriptomics,Nat.Rev.Genet.,10(1):57-63
Xie Y.,Xia H.,Liang D.,Wang Y.Z.,Liu J.,Zhuang Q.G.,and Li M.Z.,2018,Genetic diversity analysis of 25 Actinidia materials and the establishment of DNA fingerprint,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(15):5001-5007(谢玥,夏惠,梁东,王永志,刘娟,庄启国,李明章,2018,25个猕猴桃材料遗传多样性分析及DNA指纹图谱的建立,分子植物育种,16(15):5001-5007)
Yang J.,Jiang Z.W.,and Wang Y.C.,2010,Cloning and expression of dihydroflavonol 4-reductase in Actinidia chinensis var.rufopulpa,Wuhan Zhiwuxue Yanjiu(Journal of Wuhan Botanical Research),28(6):673-681(杨俊,姜正旺,王彦昌,2010,红肉猕猴桃DFR基因的克隆及表达分析,武汉植物学研究,28(6):673-681)
Zhang L.,Man Y.P.,Jiang Z.W.,and Wang Y.C.,2012,Cloning and expression of anthocyanin pathway genes,AcCHS and AcLDOX,in Actinidia chinensis,Yuanyi Xuebao(Acta Horticulturae Sinica),39(11):2124-2132(张亮,满玉萍,姜正旺,王彦昌,2012,猕猴桃花青素合成途径基因AcCHS和AcLDOX的克隆与表达分析,园艺学报,39(11):2124-2132)
Zhao Y.C.,Liu X.H.,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)