棉花色氨酸脱羧酶GhTDC基因促进烟草BY2细胞的伸长

Cotton Tryptophan Decarboxylase Gene GhTDC Promotes Cell Elongation of Tobacco BY2

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 GhTDC gene was transformed into tobacco BY2 cell to analyse the function of TDC involving in the development of cell elongation. GhTDC gene was cloned from upland cotton by using the eDNA of ovule and fiber tissue as templates of RT-PCR.The total length of eDNA of GhTDC 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 GhTDC was a hydrophilie protein,and contained typical phosphopyridoxal binding site and functional catalytic domain.Phylogenetie tree analysis revealed that the genetic relationship between GhTDC in our study and ArTDC was close Prokaryotic expression vector pET32a-GhTDC was constructed and transformed into E.eoli BI21 （DE3）.The recombinant GhTDC 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 （qRT-PCR）method indicated that the GhTDC 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 GhTDC was transformed into tobacco BY2 suspension ceils.These results provided a basis for further understanding the function of GhTDC involving in cell elongation development.