苋菜WRKY13基因克隆与生物信息学分析
|
摘要
以苋菜试管苗为材料,利用实验室构建的苋菜转录组数据库,筛选出WRKY13基因cDNA序列,对其开放阅读框(ORF)进行克隆验证和生物信息学分析。结果表明,WRKY13基因ORF长度为675 bp,编码224个氨基酸。通过生物信息学预测,该蛋白属于疏水性蛋白,有44个氨基酸位点存在磷酸化修饰;是WRKY蛋白超级家族(WRKY superfamily)的一个成员,存在1个WRKY结合位点;其二级结构由无规则卷曲结构、β折叠组成和α螺旋;蛋白系统进化树分析表明苋菜WRKY13蛋白独立形成一个分支,但与拟南芥、芜菁、烟草、葡萄聚类到一起,说明WRKY13蛋白在生物进化过程中还是比较保守的。本研究获得了苋菜WRKY13基因完整ORF,这为将来深入研究苋菜WRKY13基因功能提供了科学依据,也为研究其调控甜菜红素代谢过程的影响提供参考。
The in vitro plantlets of Amaranthus tricolor were used as the material. The c DNA sequence of WRKY13 gene was screened from transcriptome database of A. tricolor constructed in the laboratory, and the open reading frame(ORF) was used for cloning and bioinformatic analysis. The result showed that the WRKY13 ORF sequence was 675 bp in length, encoding 224 amino acids. Bioinformatics analysis showed that the protein was hydrophobic protein, and 44 amino acid sites were with phosphorylation modification. The protein was a member of WRKY superfamily with a WRKY binding site. The secondary structure consisted of random coil structure,β-sheet, and alpha helix. The analysis of protein phylogenetic tree indicated that WRKY13 protein of A. tricolor constituted a branch alone, but was associated with Arabidopsis thaliana, Brassica rapa, Nicotiana attenuata and Vitis vinifera, which indicated WRKY13 protein possesses relative conservatism during species evolution. This study has obtained the complete ORF of WRKY13 in A. tricolor, which would be helpful for further study of WRKY13 functions in A. tricolor, and might provide a reference for the study of the effects of A. tricolor WRKY13 genes on the metabolic processes of betacyanin.
The in vitro plantlets of Amaranthus tricolor were used as the material. The c DNA sequence of WRKY13 gene was screened from transcriptome database of A. tricolor constructed in the laboratory, and the open reading frame(ORF) was used for cloning and bioinformatic analysis. The result showed that the WRKY13 ORF sequence was 675 bp in length, encoding 224 amino acids. Bioinformatics analysis showed that the protein was hydrophobic protein, and 44 amino acid sites were with phosphorylation modification. The protein was a member of WRKY superfamily with a WRKY binding site. The secondary structure consisted of random coil structure,β-sheet, and alpha helix. The analysis of protein phylogenetic tree indicated that WRKY13 protein of A. tricolor constituted a branch alone, but was associated with Arabidopsis thaliana, Brassica rapa, Nicotiana attenuata and Vitis vinifera, which indicated WRKY13 protein possesses relative conservatism during species evolution. This study has obtained the complete ORF of WRKY13 in A. tricolor, which would be helpful for further study of WRKY13 functions in A. tricolor, and might provide a reference for the study of the effects of A. tricolor WRKY13 genes on the metabolic processes of betacyanin.
引文
Fu L.L.,Liu T.M.,Zhu S.Y.,Tang Q.M.,and Tang S.W.,2013,Sequence analysis of WRKY transcription factor in ramie,Zhongguo Maye Kexue(Plant Fiber Sciences in China),35(3):113-117(付莉莉,刘头明,朱四元,汤清明,唐守伟,2013,苎麻WRKY转录因子的序列分析,中国麻业科学,35(3):113-117)
Hou L.X.,Wang W.J.,and Liu X.,2013,Bioinformatics analysis of Vv WRKY13 transcription factor in Vitis vinifera L.,Beifang Yuanyi(Northern Horticul ture),(13):117-120(侯丽霞,王文杰,刘新,2013,葡萄Vv WRKY13转录因子的生物信息学分析,北方园艺,(13):117-120)
Jia C.L.,and Hou H.S.,2010,Structure of plant WRKY transcription factors and their roles in plant defense re sponses,Tianjin Nongye Kexue(Tianjin Agricultural Sciences),16(2):21-26(贾翠玲,侯和胜,2010,植物WRKY转录因子的结构特点及其在植物防卫反应中的作用,天津农业科学,16(2):21-26)
Johnson C.S.,Kolevski B.,and Smyth D.R.,2002,Transparent Testa Glabra2,a trichome and seed coat development gene of Arabidopsis,encodes a WRKY transcription factor,Plant Cell,14(6):1359-1375
Li K.,and Zhou C.J.,2014,Research progress in WRKY transcription factors in plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),50(9):1329-1335(李岢,周春江,2014,植物WRKY转录因子的研究进展,植物生理学报,50(9):1329-1335)
Li W.,2014,Gene function analysis for WRKY12 and WRKY13in Arabidopsis thaliana,Dissertation for Ph.D.,University of Science and Technology of China,Supervisor:Yu D.Q.,pp.46-51(李委,2014,拟南芥WRKY12和WRKY13的基因功能研究,博士学位论文,中国科学技术大学,导师:余迪求,pp.46-51)
Liu Z.H.,2010,Cloning of anthocyanin regulatory genes from turnip‘Tsuda’and its transformation to chrysanthemum morifolium,Thesis for M.S.,Northeast Forestry University,Supervisor:Li Y.H.,pp.1(刘振华,2010,津田芜菁花青素调控基因的克隆及转化菊花的研究,硕士学位论文,东北林业大学,导师:李玉花,pp.1)
Sun C.X.,Palmqvist S.,Olsson H.,Borén M.,Ahlandsberg S.,and Jansson C.,2003,A novel WRKY transcription factor,SUSIBA2,participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter,The Plant Cell,15(9):2076-2092
Wang C.Q.,Liu T.,and Wang B.S.,2006,Advances in betalain research in higher plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(3):302-311(王长泉,刘涛,王宝山,2006,植物甜菜素研究进展,植物学通报,23(3):302-311)
Xiao X.,2011,The cloning and function analysis of a novel WRKY transcription factors from pepper,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisor:He S.L.,pp.41(肖翔,2011,辣椒一个新的WRKY转录因子的克隆及其功能初步分析,硕士学位论文,福建农林大学,导师:何水林,pp.41)
Zhang J.,2009,Review on the function of WRKY transcription factor family,Xibei Zhiwu Xuebao(Acta Botanica BorealiOccidentalia Sinica),29(10):2137-2145(张娟,2009,WRKY转录因子功能研究进展,西北植物学报,29(10):2137-2145)
Zhang Z.L.,Xie Z.,Zou X.L.,Casaretto J.,Ho T.D.,and Shen Q.X.J.,2004,A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells,Plant Physiol.,134(4):1500-1513
Zheng X.L.,2016,Studies on physiology,biochemistryand molecular biologyon betalain synthesis in Amaranthus tricolor,Dissertation for Ph.D.,Fujian Agriculture and Forestry University,Supervisor:Lai Z.X.,pp.18-19(郑学立,2016,苋菜甜菜红素合成的生理生化与分子生物学研究,博士学位论文,福建农林大学,导师:赖钟雄,pp.18-19)
Zheng X.L.,Liu S.C.,Cheng C.Z.,Guo R.F.,Chen Y.K.,Xie L.Y.,Mao Y.Y.,Lin Y.L.,Zhang Z.H.,and Lai Z.X.,2016,Cloning and expression analysis of betalain biosynthesis genes in Amaranthus tricolor,Biotechnology Lett.,38(4):723-729
Hou L.X.,Wang W.J.,and Liu X.,2013,Bioinformatics analysis of Vv WRKY13 transcription factor in Vitis vinifera L.,Beifang Yuanyi(Northern Horticul ture),(13):117-120(侯丽霞,王文杰,刘新,2013,葡萄Vv WRKY13转录因子的生物信息学分析,北方园艺,(13):117-120)
Jia C.L.,and Hou H.S.,2010,Structure of plant WRKY transcription factors and their roles in plant defense re sponses,Tianjin Nongye Kexue(Tianjin Agricultural Sciences),16(2):21-26(贾翠玲,侯和胜,2010,植物WRKY转录因子的结构特点及其在植物防卫反应中的作用,天津农业科学,16(2):21-26)
Johnson C.S.,Kolevski B.,and Smyth D.R.,2002,Transparent Testa Glabra2,a trichome and seed coat development gene of Arabidopsis,encodes a WRKY transcription factor,Plant Cell,14(6):1359-1375
Li K.,and Zhou C.J.,2014,Research progress in WRKY transcription factors in plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),50(9):1329-1335(李岢,周春江,2014,植物WRKY转录因子的研究进展,植物生理学报,50(9):1329-1335)
Li W.,2014,Gene function analysis for WRKY12 and WRKY13in Arabidopsis thaliana,Dissertation for Ph.D.,University of Science and Technology of China,Supervisor:Yu D.Q.,pp.46-51(李委,2014,拟南芥WRKY12和WRKY13的基因功能研究,博士学位论文,中国科学技术大学,导师:余迪求,pp.46-51)
Liu Z.H.,2010,Cloning of anthocyanin regulatory genes from turnip‘Tsuda’and its transformation to chrysanthemum morifolium,Thesis for M.S.,Northeast Forestry University,Supervisor:Li Y.H.,pp.1(刘振华,2010,津田芜菁花青素调控基因的克隆及转化菊花的研究,硕士学位论文,东北林业大学,导师:李玉花,pp.1)
Sun C.X.,Palmqvist S.,Olsson H.,Borén M.,Ahlandsberg S.,and Jansson C.,2003,A novel WRKY transcription factor,SUSIBA2,participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter,The Plant Cell,15(9):2076-2092
Wang C.Q.,Liu T.,and Wang B.S.,2006,Advances in betalain research in higher plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(3):302-311(王长泉,刘涛,王宝山,2006,植物甜菜素研究进展,植物学通报,23(3):302-311)
Xiao X.,2011,The cloning and function analysis of a novel WRKY transcription factors from pepper,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisor:He S.L.,pp.41(肖翔,2011,辣椒一个新的WRKY转录因子的克隆及其功能初步分析,硕士学位论文,福建农林大学,导师:何水林,pp.41)
Zhang J.,2009,Review on the function of WRKY transcription factor family,Xibei Zhiwu Xuebao(Acta Botanica BorealiOccidentalia Sinica),29(10):2137-2145(张娟,2009,WRKY转录因子功能研究进展,西北植物学报,29(10):2137-2145)
Zhang Z.L.,Xie Z.,Zou X.L.,Casaretto J.,Ho T.D.,and Shen Q.X.J.,2004,A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells,Plant Physiol.,134(4):1500-1513
Zheng X.L.,2016,Studies on physiology,biochemistryand molecular biologyon betalain synthesis in Amaranthus tricolor,Dissertation for Ph.D.,Fujian Agriculture and Forestry University,Supervisor:Lai Z.X.,pp.18-19(郑学立,2016,苋菜甜菜红素合成的生理生化与分子生物学研究,博士学位论文,福建农林大学,导师:赖钟雄,pp.18-19)
Zheng X.L.,Liu S.C.,Cheng C.Z.,Guo R.F.,Chen Y.K.,Xie L.Y.,Mao Y.Y.,Lin Y.L.,Zhang Z.H.,and Lai Z.X.,2016,Cloning and expression analysis of betalain biosynthesis genes in Amaranthus tricolor,Biotechnology Lett.,38(4):723-729