阳春砂WRKY40的克隆、分析及原核表达
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摘要
转录因子可有效调控植物次生代谢产物的生物合成,本研究从姜科药用植物阳春砂(Amomum villosum Lour.)中克隆获得两个WRKY转录因子基因,命名为AvWRKY 40-1和AvWRKY 40-2。两个基因的编码框分别为852 bp和885 bp,分别编码283和294个氨基酸。Av WRKY 40-1和Av WRKY 40-2编码的蛋白序列一致性为75%,在GenBank中均比对上拟南芥的WRKY 40,且均含有WRKY转录因子家族所共有的WRKKYGQK七肽序列及CX5CX23HNH锌指结构域。系统发育进化树表明:Av WRKY 40-1和Av WRKY 40-2与菠萝(Ananas comosus)的AcWRKY 71亲缘关系最近,且与单子叶植物的WRKY聚为一支。两个基因在阳春砂果皮和种子团中的表达有差异,并且与阳春砂转录组中筛选出来的萜类合酶基因表达相关性也存在差异,AvWRKY 40-1表现出与萜类合酶基因更高的相关性。成功构建了两个基因的重组表达载体pDEST 17-Av WRKY 40-1和pDEST 17-Av WRKY 40-2,经诱导表达得到其融合蛋白。本研究首次克隆阳春砂的WRKY类转录因子基因并获得其融合蛋白,为后续Av WRKY 40-1和Av WRKY 40-2的功能鉴定及其对萜类的代谢调控研究提供基础。
Transcription factors can effectively regulate the biosynthesis of plant secondary metabolites. Two WRKY transcription factor genes were cloned from Amomum villosum Lour., namely AvWRKY 40-1 and AvWRKY-40-2, respectively. The coding regions of the two genes were 852 bp and 885 bp, respectively, encoding 283 and294 amino acids, respectively. The protein sequence identity of Av WRKY 40-1 and Av WRKY 40-2 was 75%.The two genes were aligned to WRKY 40 of Arabidopsis thaliana in GenBank, and they both contained a conserved WRKYGQK domain and a zinc finger structure(CX5 CX23 HNH), which are common in the WRKY transcription factors. The phylogenetic tree indicated that AvWRKY 40-1 and Av WRKY 40-2 were closely related to the AcWRKY 71 of Ananas comosus and the WRKY of monocotyledonous plants was one branch. The expression levels of AvWRKY 40-1 and AvWRKY 40-2 in pericarp and seeds were different, and their expression correlations with terpene synthase genes which were screened out from transcriptome data were different as well. Av WRKY 40-1 presented higher correlation level than Av WRKY 40-2. Meanwhile, two recombinant expression vectors, pDEST-17-Av WRKY 40-1 and pDEST 17-Av WRKY 40-2, were successfully constructed, and with the process of induction and expression, their fusion proteins were attained. In this paper, WRKY transcription factor genes and their fusion proteins were firstly cloned and obtained from the Amomum villosum Lour., which will lay a foundation for the follow-up functional characterization of Av WRKY 40-1 and Av WRKY 40-2 and the research on the metabolic regulation of terpenoids.
Transcription factors can effectively regulate the biosynthesis of plant secondary metabolites. Two WRKY transcription factor genes were cloned from Amomum villosum Lour., namely AvWRKY 40-1 and AvWRKY-40-2, respectively. The coding regions of the two genes were 852 bp and 885 bp, respectively, encoding 283 and294 amino acids, respectively. The protein sequence identity of Av WRKY 40-1 and Av WRKY 40-2 was 75%.The two genes were aligned to WRKY 40 of Arabidopsis thaliana in GenBank, and they both contained a conserved WRKYGQK domain and a zinc finger structure(CX5 CX23 HNH), which are common in the WRKY transcription factors. The phylogenetic tree indicated that AvWRKY 40-1 and Av WRKY 40-2 were closely related to the AcWRKY 71 of Ananas comosus and the WRKY of monocotyledonous plants was one branch. The expression levels of AvWRKY 40-1 and AvWRKY 40-2 in pericarp and seeds were different, and their expression correlations with terpene synthase genes which were screened out from transcriptome data were different as well. Av WRKY 40-1 presented higher correlation level than Av WRKY 40-2. Meanwhile, two recombinant expression vectors, pDEST-17-Av WRKY 40-1 and pDEST 17-Av WRKY 40-2, were successfully constructed, and with the process of induction and expression, their fusion proteins were attained. In this paper, WRKY transcription factor genes and their fusion proteins were firstly cloned and obtained from the Amomum villosum Lour., which will lay a foundation for the follow-up functional characterization of Av WRKY 40-1 and Av WRKY 40-2 and the research on the metabolic regulation of terpenoids.
引文
Chen L.P.,and He D.Y.,2010,Research advance on drought and salt resistant genes in transgenic plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(3):542-549(陈丽萍,何道一,2010,植物抗旱耐盐基因的研究进展,基因组学与应用生物学,29(3):542-549)
Chen X.L.,Yan D.,Sun L.N.,Zeng R.Z.,and Yang L.F.,2016,Cloning and expression analysis of HbWRKY 75 gene in leaf from Hevea brasiliensis,Zhiwu Shengli Xuebao(Plant Physiology Journal),3(52):250-258(陈晓丽,闫栋,孙丽娜,曾日中,杨礼富,2016,橡胶树WRKY家族转录因子HbWRKY 75基因的克隆及表达分析,植物生理学报,3(52):250-258)
Du H.,Yang W.J.,Liu L.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,Cloning and functional identification of the two MYB transcription factors GmMYBJ 6 and GmMYBJ 7in soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(7):1179-1187(杜海,杨文杰,刘蕾,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,大豆MYB转录因子基因GmMYBJ 6和G-mMYBJ 7的克隆及表达分析,作物学报,34(7):1179-1187)
He X.Y,Wang H.,Yang J.F.,Deng K.,and Wang T.,2018,RNAsequencing on Amomum villosum Lour.induced by MeJA identifies the genes of WRKY and terpene synthases involved in terpene biosynthesis,Genome,61(2):91
He X.Y.,2016,RNA sequencing on Amomum villosum Lour.induced by MeJA indentifies the genes of JA signal transduction and transcription factors involved in terpene biosynthesis,Thesis for M.S.,Guangzhou University of Chinese Medicine,Supervisor:Yang J.F.,pp.13(何雪莹,2016,基于RN-A-Seq的阳春砂JA信号转导及萜类合成相关转录因子的研究,硕士学位论文,广州中医药大学,导师:杨锦芬,pp.13)
Li J.J.,Han L.L.,Ma Y.,Yao W.J.S.,Guo J.,and Guo X.F.,2017,Cloning and expression analysis of PLWRKY 40 gene in Paeonia lactiflora Pall.,Zhiwu Shengli Xuebao(Plant Physiology Journal),53(4):609-618(李俊杰,韩璐璐,马燕,姚汪劲松,国静,郭先锋,2017,芍药转录因子Pl WRKY40的克隆及表达分析,植物生理学报,53(4):609-618)
Liu Y,He L.M.,Zhao X.T.,Liang N.S.,Li X.Y.,Liang X.,and Zhan Y.G.,2017,Cloning and expression analysis of FmWRKY 40 gene in Fraxinus mandshurica,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):833-838(刘颖,何利明,赵兴堂,梁楠松,李新宇,梁雪,詹亚光,2017,水曲柳WRKY 40基因的克隆及表达模式分析,分子植物育种,15(3):833-838)
Ma D.M.,Pu G.B.,Lei C.Y.,Ma L.Q.,Wang H.H.,Guo Y.W.,Chen J.L.,Du Z.G.,Wang H.,Li G.F.,Ye H.C.,and Liu B.Y.,2009,Isolation and characterization of AaWRKY 1,an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene,a key gene of artemisinin biosynthesis,Plant Cell Physiol.,50(12):2146-2161
MarèC.,Mazzucotelli E.,Crosatti C.,Francia E.,Stanca A.M.,and Cattivelli L.,2004,Hv-WRKY 38:a new transcription factor involved in cold-and drought-response in barley,Plant Mol.Biol.,55(3):399-416
Rushton D.L.,Tripathi P.,Rabara R.C.,Lin J.,Ringler P.,Boken A.K.,Langum T.J.,Smidt L.,Boomsma D.D.,Emme N.J.,Chen X.,Finer J.J.,Shen Q.J.,and Rushton P.J.,2012,WRKYtranscription factors:key components in abscisic acid signaling,Plant Biotechnol.J.,10(1):2-11
Sun X.L.,2011,Isolation and expression analysis of MhWRK-Y 15 in Malus hupehensis(Pamp)Rehd.,Thesis for M.S.,S-handong Agricultural University,Supervisor:Yang H.Q.,pp.4(孙晓莉,2011,平邑甜茶MhWRKY 15基因的克隆及表达分析,硕士学位论文,山东农业大学,导师:杨洪强,pp.4)
Tang L.Y.,He G.Z.,Su J.,and Xu H.H.,2012,The strategy to promote the development of industry of genuine medicinal material of Amomum villosum,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),28(8):94-99(汤丽云,何国振,苏景,徐鸿华,2012,道地春砂仁产业发展的策略研究,中国农学通报,28(8):94-99)
Tian Y.,Lu X.Y.,Peng L.S.,and Fang J.,2006,The structure and function of plant WRKY transcription factors,Yichuan(Hereditas),28(12):1607-1612(田云,卢向阳,彭丽莎,方俊,2006,植物WRKY转录因子结构特点及其生物学功能,遗传,28(12):1607-1612)
Wang Z.,Zhu Y.,Wang LL.,Liu X,Liu Y.X.,Phillips J.,and Deng X.,2009,A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase(BhGolS1)promoter,Planta,230(6):1155-1166
Xu Y.H.,Wang J.W.,Wang S.,Wang J.Y.,and Chen X.Y.,2004,Characterization of GaWRKY 1,a cotton transcription factor that regulates the sesquiterpene synthase gene(+)-δ-Cadinene synthase-A,Plant Physiol.,135:507-515
Ye W.,He X.,Wu H.Q.,Wang L.,Zhang W.M.,Fan Y.F.,Li H.H.,Liu T.M.,and Gao X.X.,2017,Identification and characterization of a nove l sesquiterpene synthase from Aquilaria sinensis:an important gene for agarwood formation,Int.J.Biol.Macromol.,108:884-892
Chen X.L.,Yan D.,Sun L.N.,Zeng R.Z.,and Yang L.F.,2016,Cloning and expression analysis of HbWRKY 75 gene in leaf from Hevea brasiliensis,Zhiwu Shengli Xuebao(Plant Physiology Journal),3(52):250-258(陈晓丽,闫栋,孙丽娜,曾日中,杨礼富,2016,橡胶树WRKY家族转录因子HbWRKY 75基因的克隆及表达分析,植物生理学报,3(52):250-258)
Du H.,Yang W.J.,Liu L.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,Cloning and functional identification of the two MYB transcription factors GmMYBJ 6 and GmMYBJ 7in soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(7):1179-1187(杜海,杨文杰,刘蕾,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,大豆MYB转录因子基因GmMYBJ 6和G-mMYBJ 7的克隆及表达分析,作物学报,34(7):1179-1187)
He X.Y,Wang H.,Yang J.F.,Deng K.,and Wang T.,2018,RNAsequencing on Amomum villosum Lour.induced by MeJA identifies the genes of WRKY and terpene synthases involved in terpene biosynthesis,Genome,61(2):91
He X.Y.,2016,RNA sequencing on Amomum villosum Lour.induced by MeJA indentifies the genes of JA signal transduction and transcription factors involved in terpene biosynthesis,Thesis for M.S.,Guangzhou University of Chinese Medicine,Supervisor:Yang J.F.,pp.13(何雪莹,2016,基于RN-A-Seq的阳春砂JA信号转导及萜类合成相关转录因子的研究,硕士学位论文,广州中医药大学,导师:杨锦芬,pp.13)
Li J.J.,Han L.L.,Ma Y.,Yao W.J.S.,Guo J.,and Guo X.F.,2017,Cloning and expression analysis of PLWRKY 40 gene in Paeonia lactiflora Pall.,Zhiwu Shengli Xuebao(Plant Physiology Journal),53(4):609-618(李俊杰,韩璐璐,马燕,姚汪劲松,国静,郭先锋,2017,芍药转录因子Pl WRKY40的克隆及表达分析,植物生理学报,53(4):609-618)
Liu Y,He L.M.,Zhao X.T.,Liang N.S.,Li X.Y.,Liang X.,and Zhan Y.G.,2017,Cloning and expression analysis of FmWRKY 40 gene in Fraxinus mandshurica,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):833-838(刘颖,何利明,赵兴堂,梁楠松,李新宇,梁雪,詹亚光,2017,水曲柳WRKY 40基因的克隆及表达模式分析,分子植物育种,15(3):833-838)
Ma D.M.,Pu G.B.,Lei C.Y.,Ma L.Q.,Wang H.H.,Guo Y.W.,Chen J.L.,Du Z.G.,Wang H.,Li G.F.,Ye H.C.,and Liu B.Y.,2009,Isolation and characterization of AaWRKY 1,an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene,a key gene of artemisinin biosynthesis,Plant Cell Physiol.,50(12):2146-2161
MarèC.,Mazzucotelli E.,Crosatti C.,Francia E.,Stanca A.M.,and Cattivelli L.,2004,Hv-WRKY 38:a new transcription factor involved in cold-and drought-response in barley,Plant Mol.Biol.,55(3):399-416
Rushton D.L.,Tripathi P.,Rabara R.C.,Lin J.,Ringler P.,Boken A.K.,Langum T.J.,Smidt L.,Boomsma D.D.,Emme N.J.,Chen X.,Finer J.J.,Shen Q.J.,and Rushton P.J.,2012,WRKYtranscription factors:key components in abscisic acid signaling,Plant Biotechnol.J.,10(1):2-11
Sun X.L.,2011,Isolation and expression analysis of MhWRK-Y 15 in Malus hupehensis(Pamp)Rehd.,Thesis for M.S.,S-handong Agricultural University,Supervisor:Yang H.Q.,pp.4(孙晓莉,2011,平邑甜茶MhWRKY 15基因的克隆及表达分析,硕士学位论文,山东农业大学,导师:杨洪强,pp.4)
Tang L.Y.,He G.Z.,Su J.,and Xu H.H.,2012,The strategy to promote the development of industry of genuine medicinal material of Amomum villosum,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),28(8):94-99(汤丽云,何国振,苏景,徐鸿华,2012,道地春砂仁产业发展的策略研究,中国农学通报,28(8):94-99)
Tian Y.,Lu X.Y.,Peng L.S.,and Fang J.,2006,The structure and function of plant WRKY transcription factors,Yichuan(Hereditas),28(12):1607-1612(田云,卢向阳,彭丽莎,方俊,2006,植物WRKY转录因子结构特点及其生物学功能,遗传,28(12):1607-1612)
Wang Z.,Zhu Y.,Wang LL.,Liu X,Liu Y.X.,Phillips J.,and Deng X.,2009,A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase(BhGolS1)promoter,Planta,230(6):1155-1166
Xu Y.H.,Wang J.W.,Wang S.,Wang J.Y.,and Chen X.Y.,2004,Characterization of GaWRKY 1,a cotton transcription factor that regulates the sesquiterpene synthase gene(+)-δ-Cadinene synthase-A,Plant Physiol.,135:507-515
Ye W.,He X.,Wu H.Q.,Wang L.,Zhang W.M.,Fan Y.F.,Li H.H.,Liu T.M.,and Gao X.X.,2017,Identification and characterization of a nove l sesquiterpene synthase from Aquilaria sinensis:an important gene for agarwood formation,Int.J.Biol.Macromol.,108:884-892