黄花蒿转录因子AaW D40的克隆
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摘要
黄花蒿(Artemisia annua L.)是菊科(Asteraceae)蒿属(Artemisia)一年生草本植物。WD40转录因子基因与植物叶片表面腺毛发育、次生代谢相关。本研究利用RACE技术从黄花蒿中克隆出Aa WD40基因(GenBank登录号:KY411922),并对其进行了生物信息学分析。AaW D40基因的cDNA全长1 498 bp,其中5'末端非翻译区为209 bp,3'末端非翻译区为183 bp。开放阅读框(ORF)的长度为1 106 bp,编码368个氨基酸残基,DNAMAN分析预测其等电点为4.64,分子量为41.29 kD。对AaW D40氨基酸序列进行同源性分析,发现其与多种植物具有较高的同源性。生物信息学分析发现AaW D40氨基酸序列含有4个典型的WD40重复区域,符合WD40基因的特征,所以命名为AaW D40。利用Swiss-Model对AaW D40蛋白进行三级结构分析,AaW D40蛋白空间结构错综复杂,与多种植物WD40空间结构域非常相似。系统发育树分析AaW D40与其他WD40均源于同一祖先,并且AaW D40与DpWDR、GhTTG4的亲缘关系最为接近。荧光实时定量PCR分析显示,AaW D40在根、茎、叶中都可以检测到表达,其中根的表达量最高,其次为叶,再次为茎。本研究为进一步研究AaW D40与黄花蒿次生代谢的关系提供了理论基础。
Artemisia annua is an annual herb plant that belongs to Asteraceae, Artemisia. Transcription factor WD40 is involved in the development of glandular hairs on the leaf surface and secondary metabolism. This study cloned AaW D40 gene(Gen Bank Accession No. KY411922) from Artemisia annua by RACE technology and analyzed its bioinformatics. The full length of cDNA in AaW D40 was 1 498 bp, of which there were 209 bp in 5' untr anslated region and 183 bp in 3' untransled region. In addition, the length of open reading frame(ORF) was 1 106 bp,encoding 368 amino acid residues with the isoelectric point of 4.64 and molecular weight about 41.29 kD through the analysis and prediction of DNAMAN. The results of homology analysis of amino acid sequence in AaW D40 showed that it had high homology with many other plants. Bioinformatics analysis suggested that amino acid sequence of AaW D40 contained 4 typical WD40-repeat motifs, which conformed to the characteristics of the WD40 gene, so it was named AaW D40. In tertiary structure analysis of AaW D40 by Swiss-Model software showed that the spatial structure of AaW D40 protein was complex, which was quite similar with the WD40 space structure domain of other plants. Phylogenetic tree analysis revealed that AaW D40 and other WD40 s originated from the same, and AaW D40 had the closest relationship with Dp WDR and Gh TTG4. Real-time PCR analysis told that the expression of AaW D40 could be detected in roots, stems and leaves, with the highest expression in roots, followed by leaves and stems in turn. This stusy provided theoretical basis for the further study of the relationship between AaW D40 and the secondary metabolism of A. annua.
Artemisia annua is an annual herb plant that belongs to Asteraceae, Artemisia. Transcription factor WD40 is involved in the development of glandular hairs on the leaf surface and secondary metabolism. This study cloned AaW D40 gene(Gen Bank Accession No. KY411922) from Artemisia annua by RACE technology and analyzed its bioinformatics. The full length of cDNA in AaW D40 was 1 498 bp, of which there were 209 bp in 5' untr anslated region and 183 bp in 3' untransled region. In addition, the length of open reading frame(ORF) was 1 106 bp,encoding 368 amino acid residues with the isoelectric point of 4.64 and molecular weight about 41.29 kD through the analysis and prediction of DNAMAN. The results of homology analysis of amino acid sequence in AaW D40 showed that it had high homology with many other plants. Bioinformatics analysis suggested that amino acid sequence of AaW D40 contained 4 typical WD40-repeat motifs, which conformed to the characteristics of the WD40 gene, so it was named AaW D40. In tertiary structure analysis of AaW D40 by Swiss-Model software showed that the spatial structure of AaW D40 protein was complex, which was quite similar with the WD40 space structure domain of other plants. Phylogenetic tree analysis revealed that AaW D40 and other WD40 s originated from the same, and AaW D40 had the closest relationship with Dp WDR and Gh TTG4. Real-time PCR analysis told that the expression of AaW D40 could be detected in roots, stems and leaves, with the highest expression in roots, followed by leaves and stems in turn. This stusy provided theoretical basis for the further study of the relationship between AaW D40 and the secondary metabolism of A. annua.
引文
Albert N.W.,Davies K.M.,Lewis D.H.,Zhang H.,Montefiori MBrendolise C.,Boase M.R.,Ngo H.,Jameson P.E.,and Schwinn K.E.,2014,A conserved network of transcriptiona activators and repressors regulates anthocyanin pigmentation in eudicots,Plant Cell,26(3):962-980
Baudry A.,Heim M.A.,Dubreucq B.,Caboche M.,Weisshaar B.and Lepiniec L.,2004,TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana,Plant J.,39(3):366-380
Ben-Simhon Z.,Judeinstein S.,Nadler-Hassar T.,Trainin T.Bar-Ya'akov I.,Borochov-Neori H.,and Holland D.,2011A pomegranate(Punica granatum L.)WD40-repeat gene is functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegra nate fruit development,Planta,234(5):865-881
Brueggemann J.,Weisshaar B.,and Sagasser M.,2010,AWD40-repeat gene from Malus×domestica is a functiona homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1,Plant Cell Rep.,29(3):285-294
Fong H.K.W.,Hurley J.B.,Hopkins R.S.,Miake-Lye R.,Johnson M.S.,Doolittle R.F.,and Simon M.I.,1986,Repetitive seg mental structure of the transducin beta subunit:homology with the CDC4 gene and identification of related m RNAs Proc.Natl.Acad.Sci.USA,83(7):2162-2166
Hernandez J.M.,Heine G.F.,Irani N.G.,Feller A.,Kim M.G.Matulnik T.,Chandler V.L.,and Grotewold E.,2004,Dif ferent mechanisms participate in the R-dependent activity o the R2R3 MYB transcription factor C1,J.Biol.Chem.,279(46):48205-48213
Humphries J.A.,Walker A.R.,Timmis J.N.,and Orford S.J.2005,Two WD-repeat genes from cotton are functional ho mologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1(TTG1)gene,Plant Mol.Biol.,57(1):67-81
Li W.,Wang B.,Wang M.,Chen M.,Yin J.M.,Kaleri G.M.Zhang R.J.,Zuo T.N.,You X.,and Yang Q.,2014,Cloning and characterization of a potato St AN11 gene involved in anthocyanin biosynthesis regulation,J.Integr.Plant Biol.56(4):364-372
Li Y.X.,Li F.,Zhou X.L.,Xie F.L.,and Li Y.G.,2014,Screening proteins interacting with the outer membrane protein Opa22in Mesorhizobium huakuii 7653R with bacterial two-hybrid library,Huazhong Nongye Daxue Xuebao(Journal of Hua zhong Agricultural University),33(2):28-33(李一星,李芳周鑫兰,谢福莉,李友国,2014,利用细菌双杂交文库筛选华癸中慢生根瘤菌7653R中与外膜蛋白Opa22互作的蛋白,华中农业大学学报,33(2):28-33)
Liu S.,Tian N.,Li J.,Huang J.N,and Liu Z.H.,2009,Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppres sion subtractive hybridization and metabolite analysis,Plan ta Med.,75(14):1542-1547
Matus J.T.,Poupin M.J.,Canon P.,Bordeu E.,Alcalde J.A.,and Arce-Johnson P.,2010,Isolation of WDR and b HLH gene related to flavonoid synthesis in grapevine(Vitis vinifera L.),Plant Mol.Biol.,72(6):607-620
Nguyen H.N.,Kim J.H.,Hyun W.Y.,Nguyen N.T.,Hong S.W.and Lee H.,2013,TTG1-mediated flavonols biosynthesi alleviates root growth inhibition in response to ABA,Plan Cell Rep.,32(4):503-514
Ouyang Y,Huang X,Lu Z,and Yao J,2012,Genomic survey expression profile and co-expression network analysis o Os WD40 family in rice.BMC Genomics,13:100-114
Pang Y.Z.,Wenger J.P.,Saathoff K.,Peel G.J.,Wen J.Q.,Huh man D.,Allen S.N.,Tang Y.H.,Cheng X.F.,Tadege M.Ratet P.,Mysore K.S.,Summer L.W.,Marks M.D.,and Dixon R.A.,2009,A WD40 repeat protein from Medicag truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome develop ment,Plant Physiol.,151(3):1114-1129
Rossi V.,Varotto S.,Locatelli S.,Lanzanova C.,Lauria M.,Zan otti E.,Hartings H.,and Motto M.,2001,The maize WD-re peat gene Zm Rb Ap1 encodes a member of the MSI/Rb Ap sub-family and is differentially expressed during endosperm development,Mol.Genet.Genomics,265(4):576-584
Stirnimann C.U.,Petsalaki E.,Russell R.B.,and Müller C.W.2010,WD40 proteins propel cellular networks,Trend Biochem Sci.,35(10):565-574
Wang Q.J.,2014,Molecular cloning of 3-hydroxy-3-methylglu taryl coenzyme A reductase and its regulation on the biosyn thesis 20-hydroxyecdysone in Cyanotis arachnoidea,Thesi for M.S.,Soochow University,Supervisor:Wang J.W.,pp.18(王秋军,2014,露水草HMGR基因的克隆分析及20-羟基蜕皮甾酮生物合成调控研究,硕士学位论文,苏州大学,导师:王剑文,pp.18)
Zhang W.,2009,The impact of triehome development related MYB genes on flower pigmentation in transgenic Tobacco Dissertation for Ph.D.,Huazhong Agricultural University Supervisor:Bao M.Z.,pp.26(张蔚,2009,与表皮毛发育相关的MYB家族基因对转基因烟草花色的影响,博士学位论文,华中农业大学,导师:包满珠,pp.26)
Baudry A.,Heim M.A.,Dubreucq B.,Caboche M.,Weisshaar B.and Lepiniec L.,2004,TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana,Plant J.,39(3):366-380
Ben-Simhon Z.,Judeinstein S.,Nadler-Hassar T.,Trainin T.Bar-Ya'akov I.,Borochov-Neori H.,and Holland D.,2011A pomegranate(Punica granatum L.)WD40-repeat gene is functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegra nate fruit development,Planta,234(5):865-881
Brueggemann J.,Weisshaar B.,and Sagasser M.,2010,AWD40-repeat gene from Malus×domestica is a functiona homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1,Plant Cell Rep.,29(3):285-294
Fong H.K.W.,Hurley J.B.,Hopkins R.S.,Miake-Lye R.,Johnson M.S.,Doolittle R.F.,and Simon M.I.,1986,Repetitive seg mental structure of the transducin beta subunit:homology with the CDC4 gene and identification of related m RNAs Proc.Natl.Acad.Sci.USA,83(7):2162-2166
Hernandez J.M.,Heine G.F.,Irani N.G.,Feller A.,Kim M.G.Matulnik T.,Chandler V.L.,and Grotewold E.,2004,Dif ferent mechanisms participate in the R-dependent activity o the R2R3 MYB transcription factor C1,J.Biol.Chem.,279(46):48205-48213
Humphries J.A.,Walker A.R.,Timmis J.N.,and Orford S.J.2005,Two WD-repeat genes from cotton are functional ho mologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1(TTG1)gene,Plant Mol.Biol.,57(1):67-81
Li W.,Wang B.,Wang M.,Chen M.,Yin J.M.,Kaleri G.M.Zhang R.J.,Zuo T.N.,You X.,and Yang Q.,2014,Cloning and characterization of a potato St AN11 gene involved in anthocyanin biosynthesis regulation,J.Integr.Plant Biol.56(4):364-372
Li Y.X.,Li F.,Zhou X.L.,Xie F.L.,and Li Y.G.,2014,Screening proteins interacting with the outer membrane protein Opa22in Mesorhizobium huakuii 7653R with bacterial two-hybrid library,Huazhong Nongye Daxue Xuebao(Journal of Hua zhong Agricultural University),33(2):28-33(李一星,李芳周鑫兰,谢福莉,李友国,2014,利用细菌双杂交文库筛选华癸中慢生根瘤菌7653R中与外膜蛋白Opa22互作的蛋白,华中农业大学学报,33(2):28-33)
Liu S.,Tian N.,Li J.,Huang J.N,and Liu Z.H.,2009,Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppres sion subtractive hybridization and metabolite analysis,Plan ta Med.,75(14):1542-1547
Matus J.T.,Poupin M.J.,Canon P.,Bordeu E.,Alcalde J.A.,and Arce-Johnson P.,2010,Isolation of WDR and b HLH gene related to flavonoid synthesis in grapevine(Vitis vinifera L.),Plant Mol.Biol.,72(6):607-620
Nguyen H.N.,Kim J.H.,Hyun W.Y.,Nguyen N.T.,Hong S.W.and Lee H.,2013,TTG1-mediated flavonols biosynthesi alleviates root growth inhibition in response to ABA,Plan Cell Rep.,32(4):503-514
Ouyang Y,Huang X,Lu Z,and Yao J,2012,Genomic survey expression profile and co-expression network analysis o Os WD40 family in rice.BMC Genomics,13:100-114
Pang Y.Z.,Wenger J.P.,Saathoff K.,Peel G.J.,Wen J.Q.,Huh man D.,Allen S.N.,Tang Y.H.,Cheng X.F.,Tadege M.Ratet P.,Mysore K.S.,Summer L.W.,Marks M.D.,and Dixon R.A.,2009,A WD40 repeat protein from Medicag truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome develop ment,Plant Physiol.,151(3):1114-1129
Rossi V.,Varotto S.,Locatelli S.,Lanzanova C.,Lauria M.,Zan otti E.,Hartings H.,and Motto M.,2001,The maize WD-re peat gene Zm Rb Ap1 encodes a member of the MSI/Rb Ap sub-family and is differentially expressed during endosperm development,Mol.Genet.Genomics,265(4):576-584
Stirnimann C.U.,Petsalaki E.,Russell R.B.,and Müller C.W.2010,WD40 proteins propel cellular networks,Trend Biochem Sci.,35(10):565-574
Wang Q.J.,2014,Molecular cloning of 3-hydroxy-3-methylglu taryl coenzyme A reductase and its regulation on the biosyn thesis 20-hydroxyecdysone in Cyanotis arachnoidea,Thesi for M.S.,Soochow University,Supervisor:Wang J.W.,pp.18(王秋军,2014,露水草HMGR基因的克隆分析及20-羟基蜕皮甾酮生物合成调控研究,硕士学位论文,苏州大学,导师:王剑文,pp.18)
Zhang W.,2009,The impact of triehome development related MYB genes on flower pigmentation in transgenic Tobacco Dissertation for Ph.D.,Huazhong Agricultural University Supervisor:Bao M.Z.,pp.26(张蔚,2009,与表皮毛发育相关的MYB家族基因对转基因烟草花色的影响,博士学位论文,华中农业大学,导师:包满珠,pp.26)