长筒石蒜类胡萝卜素裂解双加氧酶基因LlCCD4的克隆与表达
|
摘要
长筒石蒜(Lycoris longituba)是石蒜属植物,为中国特有的观赏花卉,具有极高的观赏价值也是石蒜属少有的香花品种,然而目前尚未有关于其花香方面的报道。故本研究以长筒石蒜花瓣为材料,在长筒石蒜中开展了花香基因CCD4的研究。利用课题组已获得的长筒石蒜花瓣转录组数据库,我们筛选到1个与拟南芥CCD4基因同源性最高的序列,并克隆得到了其完整的编码区,命名为LlCCD4。该基因编码区全长为1 815 bp,编码604 aa,蛋白质分子量约为66.20 kD,理论等电点为6.43,属于酸性不稳定的疏水性蛋白,且具有CCD4基因共有的保守结构域。进化分析进一步表明该基因为CCDs基因家族中的CCD4成员,与单子叶植物番红花中的CCD4基因具有较高的同源性。实时荧光定量分析证实该基因在长筒石蒜的盛花期即长筒石蒜花瓣最香的时期表达量最高。本研究将有助于揭示长筒石蒜花香形成的分子机理,并为石蒜属植物花香改良提供基因资源。
Lycoris longituba(Lycoris Herb) is a unique ornamental flower in China, which has very high ornamental value as a rare fragrant flower variety of Lycoris. However, there is no report about its fragrance at present.Therefore, in this research, the floral gene CCD4 was studied with the petals of Lycoris longituba as materials.Based on the transcriptome database of L. longituba petals obtained by the research team, a sequence with the highest homology to Arabidopsis thaliana CCD4 gene was screened and its complete coding region was cloned,named as Ll CCD4. The full length of LlCCD4 coding region was 1 815 bp, encoding 604 aa, and its protein molecular weight was approximately 66.20 kD, the theoretical isoelectric point was 6.43. It was an acid-labile hydrophobic protein that had a conserved domain shared by CCD4 gene. The evolution analysis further showed that LlCCD4 was a member of CCD4 in CCDs gene family, which had a high homology with CCD4 gene in monocotyledonous Crocus sativus. The q RT-PCR analysis showed that the expression of LlCCD4 was the highest at the blooming stage of L. longituba, that is, the strongest fragrance release stage. This study would help to reveal the molecular mechanism of the floral formation of Lycoris longituba and to provide genetic resources for the improvement of floral aroma of Lycoris.
Lycoris longituba(Lycoris Herb) is a unique ornamental flower in China, which has very high ornamental value as a rare fragrant flower variety of Lycoris. However, there is no report about its fragrance at present.Therefore, in this research, the floral gene CCD4 was studied with the petals of Lycoris longituba as materials.Based on the transcriptome database of L. longituba petals obtained by the research team, a sequence with the highest homology to Arabidopsis thaliana CCD4 gene was screened and its complete coding region was cloned,named as Ll CCD4. The full length of LlCCD4 coding region was 1 815 bp, encoding 604 aa, and its protein molecular weight was approximately 66.20 kD, the theoretical isoelectric point was 6.43. It was an acid-labile hydrophobic protein that had a conserved domain shared by CCD4 gene. The evolution analysis further showed that LlCCD4 was a member of CCD4 in CCDs gene family, which had a high homology with CCD4 gene in monocotyledonous Crocus sativus. The q RT-PCR analysis showed that the expression of LlCCD4 was the highest at the blooming stage of L. longituba, that is, the strongest fragrance release stage. This study would help to reveal the molecular mechanism of the floral formation of Lycoris longituba and to provide genetic resources for the improvement of floral aroma of Lycoris.
引文
Auldridge M.E.,McCarty D.R.,and Klee H.J.,2006,Plant carotenoid cleavage oxygenases and their apocarotenoid products,Curr.Opin.Plant Biol.,9(3):315-321
Baldermann S.,Kato M.,Kurosawa M.,Kurobayashi Y.,Fujita A.Fleischmann P.,and Watanabe N.,2010,Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour.,J.Exp.Bot.,61(11):2967-2977
Brandi F.,Bar E.,Mourgues F.,Horváth G.,Turcsi E.,Giuliano G.,Liverani A.,Tartarini S.,Lewinsohn E.,and Rosati C.,2011,Study of'Redhaven'peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism,BMC Plant Biol.,11:24
Cui S.J.,He Q.L.,Chen Y.,and Huang M.R.,2011,Evaluation of suitable reference genes for gene expression studies in Lyycoris longituba,J.Genet.,90(3):503-506
He Q.,Shen Y.,Wang M.,Huang M.,Yang R.,Zhu S,Wang L.,Xu Y.,and Wu R.,2011,Natural variation in petal color in Lycoris longituba revealed by anthocyanin components,PLoSOne,6(8):e22098
He Q.L.,2009,The basic relative study on molecular biology of color variation in Lycoris longituba,Dissertation for Ph.D.,Nanjing Forestry University,Supervisor:Wang M.M.,and Huang M.R.,pp.3-4(何秋伶,2009,长筒石蒜花色变异的分子基础,博士学位论文,南京林业大学,导师:王明麻,黄敏仁,pp.3-4)
Huang F.C.,Molnár P.,and Schwab W.,2009,Cloning and functional characterization of carotenoid cleavage dioxygenase 4genes,J.Exp.Bot.,60(11):3011-3022
Ibdah M.,Azulay Y.,Portnoy V.,Wasserman B.,Bar E.,Meir A.,Burger Y.,Hirschberg J.,Schaffer A.A.,Katzir N.,Tadmor Y.,and Lewinsohn E.,2006,Functional characterization of Cm CCD1,a carotenoid cleavage dioxygenase from melon,Phytochemistry,67(15):1579-1589
Liu K.,Tang C.F.,Zhou S.B.,Wang Y.P.,Zhang D.,Wu G.W.,and Chang L.L.,2012,Comparison of the photosynthetic characteristics of four Lycoris species with leaf appearing in autumn under field conditions,Photosynthetica,50(4):570-576
Mu H.N.,Sun T.Z.,Xu C.,Li H.G.,Yang X.L.,and Wang L.G.,2016,Gene clone and expression analysis of 4-coumarate-co a ligase in sweet Osmanthus(Osmanthus fragrans Lour.),Fenzi Z-hiwu Yuzhong(Molecular Plant Breeding),14(3):536-541(母洪娜,孙陶泽,徐晨,李火根,杨秀莲,王良桂,2016,桂花(Osmanthus fragrans Lour.)4-香豆酸辅酶A连接酶(4CL)基因克隆与表达分析,分子植物育种,14(3):536-541)
Rubio A.,Rambla J.L.,Santaella M.,Gómez M.D.,Orzaez D.,Granell A.,and Gómez-Gómez L.,2008,Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved inβ-ionone release,J.Biol.Chem.,283(36):24816-24825
Simkin A.J.,Underwood B.A.,Auldridge M.,Loucas H.M.,Shibuya K.,Schmelz E.,Clark D.G.,and Klee H.J.,2004,Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission ofβ-Ionone,a fragrance volatile of petunia flowers,Plant Physiol.,136(3):3504-3514
Tan B.C.,Joseph L.M.,Deng W.T.,Liu L.,Li Q.B.,Cline K.,and McCarty D.R.,2003,Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family Plant J.,35(1):44-56
Wang B.,Han L.M.,Hua W.P.,and Chen C.,2015,Bioinformatics and tissue expression pattern analysis on the coumarate--3-hydroxylase gene(C3H)in Salvia miltiorrhiza Bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):813-820(王斌,韩立敏,化文平,陈尘,2015丹参香豆酸-3-羟化酶基因(SmC3H)的生物信息学及其组织表达模式分析,基因组学与应用生物学,34(4)813-820)
Wang R.,Xu S.,Jiang Y.,Jiang J.,Li X.,Liang L.,He J.,Peng F.,and Xia B.,2013,De novo sequence assembly and characterization of Lycoris aurea transcriptome using GS FLX titanium platform of 454 pyrosequencing,PLoS One,8(4):e60449
Zhang X.S.,Pei J.J.,Zhao L.G.,Tang F.,Fang X.Y.,and Xie J.C.,2016,Overexpression and characterization of CCD4 from Osmanthus fragrans andβ-ionone biosynthesis fromβ-carotene in vitro,J.Mol.Catal.B-Enzym.,134:105-114
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)
Baldermann S.,Kato M.,Kurosawa M.,Kurobayashi Y.,Fujita A.Fleischmann P.,and Watanabe N.,2010,Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour.,J.Exp.Bot.,61(11):2967-2977
Brandi F.,Bar E.,Mourgues F.,Horváth G.,Turcsi E.,Giuliano G.,Liverani A.,Tartarini S.,Lewinsohn E.,and Rosati C.,2011,Study of'Redhaven'peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism,BMC Plant Biol.,11:24
Cui S.J.,He Q.L.,Chen Y.,and Huang M.R.,2011,Evaluation of suitable reference genes for gene expression studies in Lyycoris longituba,J.Genet.,90(3):503-506
He Q.,Shen Y.,Wang M.,Huang M.,Yang R.,Zhu S,Wang L.,Xu Y.,and Wu R.,2011,Natural variation in petal color in Lycoris longituba revealed by anthocyanin components,PLoSOne,6(8):e22098
He Q.L.,2009,The basic relative study on molecular biology of color variation in Lycoris longituba,Dissertation for Ph.D.,Nanjing Forestry University,Supervisor:Wang M.M.,and Huang M.R.,pp.3-4(何秋伶,2009,长筒石蒜花色变异的分子基础,博士学位论文,南京林业大学,导师:王明麻,黄敏仁,pp.3-4)
Huang F.C.,Molnár P.,and Schwab W.,2009,Cloning and functional characterization of carotenoid cleavage dioxygenase 4genes,J.Exp.Bot.,60(11):3011-3022
Ibdah M.,Azulay Y.,Portnoy V.,Wasserman B.,Bar E.,Meir A.,Burger Y.,Hirschberg J.,Schaffer A.A.,Katzir N.,Tadmor Y.,and Lewinsohn E.,2006,Functional characterization of Cm CCD1,a carotenoid cleavage dioxygenase from melon,Phytochemistry,67(15):1579-1589
Liu K.,Tang C.F.,Zhou S.B.,Wang Y.P.,Zhang D.,Wu G.W.,and Chang L.L.,2012,Comparison of the photosynthetic characteristics of four Lycoris species with leaf appearing in autumn under field conditions,Photosynthetica,50(4):570-576
Mu H.N.,Sun T.Z.,Xu C.,Li H.G.,Yang X.L.,and Wang L.G.,2016,Gene clone and expression analysis of 4-coumarate-co a ligase in sweet Osmanthus(Osmanthus fragrans Lour.),Fenzi Z-hiwu Yuzhong(Molecular Plant Breeding),14(3):536-541(母洪娜,孙陶泽,徐晨,李火根,杨秀莲,王良桂,2016,桂花(Osmanthus fragrans Lour.)4-香豆酸辅酶A连接酶(4CL)基因克隆与表达分析,分子植物育种,14(3):536-541)
Rubio A.,Rambla J.L.,Santaella M.,Gómez M.D.,Orzaez D.,Granell A.,and Gómez-Gómez L.,2008,Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved inβ-ionone release,J.Biol.Chem.,283(36):24816-24825
Simkin A.J.,Underwood B.A.,Auldridge M.,Loucas H.M.,Shibuya K.,Schmelz E.,Clark D.G.,and Klee H.J.,2004,Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission ofβ-Ionone,a fragrance volatile of petunia flowers,Plant Physiol.,136(3):3504-3514
Tan B.C.,Joseph L.M.,Deng W.T.,Liu L.,Li Q.B.,Cline K.,and McCarty D.R.,2003,Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family Plant J.,35(1):44-56
Wang B.,Han L.M.,Hua W.P.,and Chen C.,2015,Bioinformatics and tissue expression pattern analysis on the coumarate--3-hydroxylase gene(C3H)in Salvia miltiorrhiza Bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):813-820(王斌,韩立敏,化文平,陈尘,2015丹参香豆酸-3-羟化酶基因(SmC3H)的生物信息学及其组织表达模式分析,基因组学与应用生物学,34(4)813-820)
Wang R.,Xu S.,Jiang Y.,Jiang J.,Li X.,Liang L.,He J.,Peng F.,and Xia B.,2013,De novo sequence assembly and characterization of Lycoris aurea transcriptome using GS FLX titanium platform of 454 pyrosequencing,PLoS One,8(4):e60449
Zhang X.S.,Pei J.J.,Zhao L.G.,Tang F.,Fang X.Y.,and Xie J.C.,2016,Overexpression and characterization of CCD4 from Osmanthus fragrans andβ-ionone biosynthesis fromβ-carotene in vitro,J.Mol.Catal.B-Enzym.,134:105-114
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)