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赖氨酸脱羧酶(lysine decarboxylase,LDC)是生物碱合成第一步所需的关键酶。为研究LDC基因在烟草中特性和功能,本研究采用同源克隆和RT-PCR方法从栽培烟草‘K326’中克隆得到一个LDC基因,命名为Nt LDC1,Gen Bank登录号为KU507075。序列分析表明烟草Nt LDC1基因ORF全长666 bp,编码221个氨基酸的蛋白,相对分子质量为24 264.9 Da,等电点为5.43。不同植物中LDC蛋白较为保守,Nt LDC1与番茄和马铃薯的LDC蛋白高度相似,进化分析表明Nt LDC1与番茄中LDC的亲缘关系最近。利用实时荧光定量PCR对Nt LDC1基因进行组织表达分析,结果显示Nt LDC1基因在烟草根、茎、叶、花中均有表达,在叶片中的表达水平最高。低温可以诱导Nt LDC1基因的表达,在低温处理8 h基因的表达量达到最高,表明Nt LDC1可能在烟草低温胁迫应答中发挥作用。
Lysine decarboxylase (LDC) is the key enzyme required for the first step of alkaloid synthesis. In order to study the characteristics and functions of LDC genes in tobacco, a LDC gene was cloned from tobacco “K326” by homologous cloning and RT-PCR, named as Nt LDC1, Gen Bank Accession No. KU507075. Sequence analysis showed that the ORF of tobacco Nt LDC1 gene was 666 bp in length, encoding a 221 amino acid protein with a relative molecular mass of 24 264.9 Da and an isoelectric point of 5.43. LDC protein is more conservative in different plants. Nt LDC1 is highly similar to LDC protein in tomato and potato. Phylogenetic analysis shows that Nt LDC1 is the most closely related to LDC in tomato. Tissue expression analysis of Nt LDC1 gene by real-time fluorescence quantitative PCR showed that Nt LDC1 gene was expressed in tobacco roots, stems, leaves, flowers and the highest expression level in leaves. Low temperature could induce the expression of Nt LDC1 gene, which reached the highest level at 8 h after cold treatment, indicating that Nt LDC1 may play a role in the response to low temperature stress in tobacco.