牛大力漆酶基因CsLAC17的克隆与载体构建
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摘要
为研究漆酶在牛大力生长发育过程中的生物学功能,本实验以牛大力叶片为材料,从反转录PCR获得的cDNA中扩增出漆酶基因CsLAC17全长。序列分析表明,CsLAC17 c DNA全长为2 010 bp,开放阅读框大小为1 755 bp,编码一个由584个氨基酸组成的蛋白质。结构域分析表明,Cs LAC17蛋白的保守结构域具有漆酶典型结构域的特征——铜离子结合域(Cu-oxidase和Cu-oxidase-2)。同源序列分析表明,Cs LAC17蛋白序列与绿豆(Vigna radiata var. radiata)和野生大豆(Glycine soja)同源性为88%、藜豆(Mucuna pruriens)87%、蒺藜苜蓿(Medicago truncatula) 85%。组织特异性表达分析显示,CsLAC17在茎中表达量最高,叶中表达量次之,根中较少。此外,进一步构建了pBI121-CsLAC17过表达载体并转入农杆菌。本研究为日后CsLAC17基因的功能验证以及为牛大力开展分子生物学研究提供帮助。
In this study, the cDNA of LACcase gene Cs LAC17 was amplified by RT-PCR from leaf of Millettia specisoa Champ. Squencing analysis revealed that the full length of Cs LAC17 is 2 010 bp which contains 1 755 bp open reading frame(ORF), and coding a protein consisted by 584 amino acid. Structural domain analysis revealed that the deduced CsLAC17 protein contains copper binding domains(Cu-oxidase and Cu-oxidase-2) which shared the typical characteristics of the LACtase family. Homologous sequence analysis indicated that CsLAC17 protein sequence was 88 percent homologous to Vigna radiata var. radiata and Glycine soja, 87 percent to Mucuna pruriens, and 85 percent to Medicago truncatula. qRT-PCR analysis in different tissues showed that CsLAC17 exhibited the highest expression levels in roots, followed by leaf and less in root. Furthermore, a pBI121-CsLAC17 overexpression vector was constructed, and the plasmid was transformed into Agrobacterium tumefacien. The experiment results will be helpful to the biological function verification of CsLAC17 gene and and to the molecular biology research of C. speciosa.
In this study, the cDNA of LACcase gene Cs LAC17 was amplified by RT-PCR from leaf of Millettia specisoa Champ. Squencing analysis revealed that the full length of Cs LAC17 is 2 010 bp which contains 1 755 bp open reading frame(ORF), and coding a protein consisted by 584 amino acid. Structural domain analysis revealed that the deduced CsLAC17 protein contains copper binding domains(Cu-oxidase and Cu-oxidase-2) which shared the typical characteristics of the LACtase family. Homologous sequence analysis indicated that CsLAC17 protein sequence was 88 percent homologous to Vigna radiata var. radiata and Glycine soja, 87 percent to Mucuna pruriens, and 85 percent to Medicago truncatula. qRT-PCR analysis in different tissues showed that CsLAC17 exhibited the highest expression levels in roots, followed by leaf and less in root. Furthermore, a pBI121-CsLAC17 overexpression vector was constructed, and the plasmid was transformed into Agrobacterium tumefacien. The experiment results will be helpful to the biological function verification of CsLAC17 gene and and to the molecular biology research of C. speciosa.
引文
Edlund A.F.,Swanson R.,and Preuss D.,2004,Pollen and stigma structure and function:the role of diversity in pollination,Plant Cell,16(S):84-97
Fu M.Q.,Xiao G.S.,Xu Y.J.,Wu J.J.,Chen Y.L.,and Qiu S.X.,2016,Chemical constituents from roots of Millettia speciosa,Chinese Herbal Medicines,8(4):385-389
Gorbacheva M.A.,Shumakovich G.P.,Morozova O.V.,Strel'tsov A.V.,Zaitseva E.A.,and Shleev S.V.,2008,Comparative study of biocatalytic reactions of high and low redox potential fungal and plant LACcases in homogeneous and heterogeneous reactions,Moscow University Chemistry Bulletin,63(2):94-98
Hu Q.N.,Luo C.,Zhang Q.L.,and Luo Z.R.,2013,Isolation and characterization of a LACcase gene potentially involved in proanthocyanidin polymerization in oriental persimmon(Diospyros kaki Thunb.)fruit,Mol.Biol.Rep.,40(4):2809-2820
Li L.,and Steffens J.C.,2002,Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance,Planta,215(2):239-247
Luo Y.C.,Zhou H.,Li Y.,Chen J.Y.,Yang J.H.,Chen Y.Q.,and Qu L.H.,2006,Rice embryogenic calli express a unique set of microRNAs,suggesting regulatory roles of microRNAs in plant post-embryogenic development,FEBS Lett.,580(21):5111-5116
Mate D.M.,and Alcalde M.,2015,Laccase engineering:from rational design to directed evolution,Biotechnol.Adv.,33(1):25-40
Pan Y.N.,Zhang X.J.,Yu W.H.,Meng P.,Deng H.Y.,and Li T.,2016,Study on plant growth characteristics and chemical removal of flower bud of Millettia specisoa Champ.,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),29(3):678-682(潘颖南,张向军,庾韦花,蒙平,邓海燕,李婷,2016,牛大力植株生长特性及化学去蕾研究,西南农业学报,29(3):678-682)
Pardo I.,and Camarero S.,2015,Laccase engineering by rational and evolutionary design,Cell.Mol.Life Sci.,72(5):897-910
Pourcel L.,Routaboul J.M.,and Kerhoas L.,2005,TRANSPAR-ENT TESTA10 encodes a Laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat,Plant Cell,17(11):2966-2980
Serge B.,Demont-Caulet N.,Pollet B.,Bidzinski P.,Cezard L.,Bris P.L.,Borrega N.,Herve J.,Blondet E.,Balzergue S.,Lapierre C.,and Jouanin L.,2011,Disruption of LACCASE4and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems,Plant Cell,23(3):1124-1137
Turlapati P.V.,Kim K.W.,Davin L.B.,and Lewis N.G.,2011,The Laccase multigene family in Arabidopsis thaliana:Towards addressing the mystery of their gene function(s),Planta,233(3):439-470
Zhang S.C.,Zhang Y.P.,and Wang X.J.,2012,Arabidopsis thaliana laccase gene AtLAC2 regulates plant growth and development,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(6):597-604(张盛春,张玉平,王小菁,2012,拟南芥漆酶基因AtLAC2调控植物生长发育的研究,植物生理学报,48(6):597-604)
Zhao Q.,Jin N.,Chen F.,Yin Y.B.,Fu C.X.,Yun J.F.,Shao H.,Wang X.Q.,Wang Z.Y.,and Dixona R.A.,2013,LACCASEis necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis,Plant Cell,25(10):3976-3987
Zhao X.Y.,Pang M.L.,Zhao Q.,Ren Y.R.,Hao Y.J.,and You C.X.,2015,Cloning and expression analysis of tomato LeLACmi R397gene,Yuanyi Xuebao(Acta Horticulturae Sinica),42(7):1285-1298(赵先炎,庞明利,赵强,任怡然,郝玉金,由春香,2015,番茄漆酶基因LeLACmi R397的克隆与表达分析,园艺学报,42(7):1285-1298)
Fu M.Q.,Xiao G.S.,Xu Y.J.,Wu J.J.,Chen Y.L.,and Qiu S.X.,2016,Chemical constituents from roots of Millettia speciosa,Chinese Herbal Medicines,8(4):385-389
Gorbacheva M.A.,Shumakovich G.P.,Morozova O.V.,Strel'tsov A.V.,Zaitseva E.A.,and Shleev S.V.,2008,Comparative study of biocatalytic reactions of high and low redox potential fungal and plant LACcases in homogeneous and heterogeneous reactions,Moscow University Chemistry Bulletin,63(2):94-98
Hu Q.N.,Luo C.,Zhang Q.L.,and Luo Z.R.,2013,Isolation and characterization of a LACcase gene potentially involved in proanthocyanidin polymerization in oriental persimmon(Diospyros kaki Thunb.)fruit,Mol.Biol.Rep.,40(4):2809-2820
Li L.,and Steffens J.C.,2002,Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance,Planta,215(2):239-247
Luo Y.C.,Zhou H.,Li Y.,Chen J.Y.,Yang J.H.,Chen Y.Q.,and Qu L.H.,2006,Rice embryogenic calli express a unique set of microRNAs,suggesting regulatory roles of microRNAs in plant post-embryogenic development,FEBS Lett.,580(21):5111-5116
Mate D.M.,and Alcalde M.,2015,Laccase engineering:from rational design to directed evolution,Biotechnol.Adv.,33(1):25-40
Pan Y.N.,Zhang X.J.,Yu W.H.,Meng P.,Deng H.Y.,and Li T.,2016,Study on plant growth characteristics and chemical removal of flower bud of Millettia specisoa Champ.,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),29(3):678-682(潘颖南,张向军,庾韦花,蒙平,邓海燕,李婷,2016,牛大力植株生长特性及化学去蕾研究,西南农业学报,29(3):678-682)
Pardo I.,and Camarero S.,2015,Laccase engineering by rational and evolutionary design,Cell.Mol.Life Sci.,72(5):897-910
Pourcel L.,Routaboul J.M.,and Kerhoas L.,2005,TRANSPAR-ENT TESTA10 encodes a Laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat,Plant Cell,17(11):2966-2980
Serge B.,Demont-Caulet N.,Pollet B.,Bidzinski P.,Cezard L.,Bris P.L.,Borrega N.,Herve J.,Blondet E.,Balzergue S.,Lapierre C.,and Jouanin L.,2011,Disruption of LACCASE4and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems,Plant Cell,23(3):1124-1137
Turlapati P.V.,Kim K.W.,Davin L.B.,and Lewis N.G.,2011,The Laccase multigene family in Arabidopsis thaliana:Towards addressing the mystery of their gene function(s),Planta,233(3):439-470
Zhang S.C.,Zhang Y.P.,and Wang X.J.,2012,Arabidopsis thaliana laccase gene AtLAC2 regulates plant growth and development,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(6):597-604(张盛春,张玉平,王小菁,2012,拟南芥漆酶基因AtLAC2调控植物生长发育的研究,植物生理学报,48(6):597-604)
Zhao Q.,Jin N.,Chen F.,Yin Y.B.,Fu C.X.,Yun J.F.,Shao H.,Wang X.Q.,Wang Z.Y.,and Dixona R.A.,2013,LACCASEis necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis,Plant Cell,25(10):3976-3987
Zhao X.Y.,Pang M.L.,Zhao Q.,Ren Y.R.,Hao Y.J.,and You C.X.,2015,Cloning and expression analysis of tomato LeLACmi R397gene,Yuanyi Xuebao(Acta Horticulturae Sinica),42(7):1285-1298(赵先炎,庞明利,赵强,任怡然,郝玉金,由春香,2015,番茄漆酶基因LeLACmi R397的克隆与表达分析,园艺学报,42(7):1285-1298)