棉花色氨酸脱羧酶GhTDC基因促进烟草BY2细胞的伸长
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摘要
色氨酸脱羧酶(Tryptophan decarboxylase,TDC)在生长素(Indoleacetic acid,IAA)合成和植物器官发育等过程中发挥重要作用,其参与纤维发育的研究现有报道。本研究将陆地棉Gh TDC基因转化烟草BY2悬浮细胞,分析TDC在细胞伸长发育过程中的功能。本研究采用RT-PCR方法从陆地棉胚珠和纤维组织中克隆得到Gh TDC基因,该基因的c DNA全长包含完整开放读码框为1491 bp,编码497个氨基酸的多肽,理论分子质量为54.67 ku。生物信息学分析结果表明,Gh TDC蛋白是一个亲水性蛋白,其含有磷酸吡哆醛结合位点和催化结构域。进化树分析结果表明,棉花Gh TDC与黑升麻Ar TDC的亲缘关系上较近。构建原核表达载体p ET32a-Gh TDC并转化大肠杆菌BL21(DE3)诱导表达,获得分子质量约为54 ku的重组蛋白,重组蛋白具有较高的酶催化活力。q RT-PCR结果分析结果表明,Gh TDC基因在纤维突起时期的开花前3 d和纤维快速伸长发育时期的开花后5-15 d表达丰度较高。将Gh TDC转基因转化烟草BY2悬浮细胞,烟草悬浮细胞的伸长发育获得了显著促进。本研究结果为研究Gh TDC基因在细胞伸长发育过程中的重要调控作用提供了参考。
Tryptophan decarboxylase(TDC)plays important roles in Indoleacetic acid(IAA)biosynthesis and plant organ development,and its participation in the in the process of fiber development has been reported previously.Here,cotton Gh TDC gene was transformed into tobacco BY2 cell to analyse the function of TDC involving in the development of cell elongation. Gh TDC gene was cloned from upland cotton by using the c DNA of ovule and fiber tissue as templates of RT-PCR.The total length of c DNA of Gh TDC gene contains 1491 bp open reading frame(ORF)and codes a protein of 497 amino acid residues with theoretical molecular weight of 54.67 ku.Bioinformatics analysis showed that Gh TDC was a hydrophilic protein,and contained typical phosphopyridoxal binding site and functional catalytic domain.Phylogenetic tree analysis revealed that the genetic relationship between Gh TDC in our study and Ar TDC was close Prokaryotic expression vector p ET32a-Gh TDC was constructed and transformed into E.coli BL21(DE3).The recombinant Gh TDC protein with a molecular weight of about 54 ku was obtained after induction by IPTG,and the recombinant protein had a high enzyme activity.Gene expression analysis by using quantitative real time RT- PCR(q RT-PCR)method indicated that the Gh TDC gene expressed highly at-3 day post anthesis(dpa) of fiber initiation stage and 5 to 15 dpa of fiber fast elongation state.The growth of tobacco suspension cell was significantly promoted when Gh TDC was transformed into tobacco BY2 suspension cells.These results provided a basis for further understanding the function of Gh TDC involving in cell elongation development.
Tryptophan decarboxylase(TDC)plays important roles in Indoleacetic acid(IAA)biosynthesis and plant organ development,and its participation in the in the process of fiber development has been reported previously.Here,cotton Gh TDC gene was transformed into tobacco BY2 cell to analyse the function of TDC involving in the development of cell elongation. Gh TDC gene was cloned from upland cotton by using the c DNA of ovule and fiber tissue as templates of RT-PCR.The total length of c DNA of Gh TDC gene contains 1491 bp open reading frame(ORF)and codes a protein of 497 amino acid residues with theoretical molecular weight of 54.67 ku.Bioinformatics analysis showed that Gh TDC was a hydrophilic protein,and contained typical phosphopyridoxal binding site and functional catalytic domain.Phylogenetic tree analysis revealed that the genetic relationship between Gh TDC in our study and Ar TDC was close Prokaryotic expression vector p ET32a-Gh TDC was constructed and transformed into E.coli BL21(DE3).The recombinant Gh TDC protein with a molecular weight of about 54 ku was obtained after induction by IPTG,and the recombinant protein had a high enzyme activity.Gene expression analysis by using quantitative real time RT- PCR(q RT-PCR)method indicated that the Gh TDC gene expressed highly at-3 day post anthesis(dpa) of fiber initiation stage and 5 to 15 dpa of fiber fast elongation state.The growth of tobacco suspension cell was significantly promoted when Gh TDC was transformed into tobacco BY2 suspension cells.These results provided a basis for further understanding the function of Gh TDC involving in cell elongation development.
引文
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[2]Facchini P J,Huber-allanach K L,Tari L W.Plant aromatic l-amino acid decarboxylases:evolution,biochemistry,regulation,and metabolic engineering applications[J].Phytochemistry,2000,54:121-138.
[3]孙世芹,阎秀峰.喜树幼苗中喜树碱及其代谢相关酶类的分布[J].中草药,2008,39(8):1231-1234.Sun Shiqin,Yan Xiufeng.Distribution of camptothecin and itsmetabolism-related enzymes in Camptotheca acum inata seedlings[J].Chinese Traditional and Herbal Drugs,2008,39(8):1231-1234.
[4]Dai X H,Mashiguchi K,Chen Q,et al.The biochemical mechanism of auxin biosynthesis by an arabidopsis YUCCA flavin-containing Monooxygenase[J].Biol Chem,2013,288:1448-1457.
[5]Liu Wanhong,Chen Rong,Chen Min,et al.Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata[J].Planta,2012,236:239-250.
[6]夏兰芹,郭三堆.棉花RNA的快速提取方法[J].棉花学报,2000,12(4):205-207.Xia Lanqin,GUO Sandui.A method of RNA fast extracing in cotton tissues[J].Acta Gossypii Sinica Acta Gossypii Sinica,2000,12(4):205-207.
[7]田大鹏,葛娟,石峰,等.棉花Gh DHAR2基因的克隆、功能序列分析及原核表达[J].生物技术通报,2012(7):65-69.Tian Dapeng,Ge Juan,Shi Feng,et al.Cloning,functional sequence analysis and prokaryotic expression of cotton Gh DHAR2 c DNA[J].Biotechnology Bulletin,2012(7):65-69.
[8]李学宁,杜军伟,李鸿彬.棉花脱氢抗坏血酸还原酶基因的克隆、原核表达与纯化[J].石河子大学学报:自然科学版,2010,28(5):542-545.Li Xuening,Du Junwei,Li Hongbin.Cloning,prokaryotic expression and purification of a cotton dehydroascorbate reductase cene[J].Journal of Shihezi University:Natural Science,2010,28(5):542-545.
[9]王芳,王斐,孙辉,等.棉花Gh DHAR3基因克隆、功能序列分析及烟草的遗传转化[J].西北农业学报,2011,20(5):88-93.Wang Fang,Wang Fei,Sun Hui,et al.,Cloning and sequence analysis of a cotton DHAR3 c DNA and genetic tranansformation of tobacco[J].Acta Agriculturae Borealioccidentalis Sinica,2011,20(5):88-93.
[10]Aerts R J,Alarco A M,De L V.Auxins induce tryptophan decarboxylase activity in radicles of Catharanthus seedlings[J].Plant Physiol,1992,100:1014-1019.
[11]Pennings E J,Hegger I,Duine J A,et al.Assay of tryptophan decarboxylase from Catharanthus roseusplant cel cultures by high-performance liquid chromatography[J].Anal Bio Chem,1987,165:133-136.
[12]唐忠海,饶力群,李兰岚.长春花愈伤组织的诱导及限速酶活性的研究[J].天然产物研究与开发,2006,18(4):596-598.Tang Zhonghai,Rao Liqun,Li Lanlan.Callus initiation and the activity of key enzyme in Catharanthus roseus(L.)G.Don.[J]Nat Prod Res Dev,2006,18(4):596-598.
[13]Liu W,Chen R,Chen M,et al.Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata[J].Planta,2012,236:239-250.
[14]Liu N,Lin Z,Guan L,et al.Antioxidant enzymes regulate reactive oxygen species during pod elongation in Pisum sativum and Brassica chinensis[J].PLo S ONE,2014,9(2):e87588.
[15]Liu N,Lin Z F.Reactive oxygen species and alternative respiration in the developing flowers of two subtropical woody plants[J].J Plant Growth Regul,2013,32:83-91.
[16]Li H B,Qin Y M,Pang Y,et al.A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development[J].New Phytologist,2007,175:462-471.
[17]Foreman J,Demidchik V,Bothwell J,et al.Reactive oxygen species produced by NADPH oxidase regulate plant cell growth[J].Nature,2003,422(6930):442-446.
[18]Mittler R,Vanderauwera S,Gollery M F,et al.Reactive oxygen gene network of plants[J].Trends Plant Sci,2004,9(10):490-498.
[19]Schopfer P.Hydroxyl radical-induced cell-wall loosening in vitro and in vivo:implications for the control of elongation growth[J].Plant J,2001,28(6):679-688.