烟草赖氨酸脱羧酶基因NtLDC1的克隆和表达分析

Cloning and expression analysis of lysine decarboxylase gene NtLDC1 from Nicotiana tabacum

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 in the first step of alkaloid synthesis. In order to study the characteristics and fuction of LDC in tobacco, we cloned a LDC gene from Nicotiana tabacum ‘K326＇ by RT-PCR technology, naming Nt LDC1, Gen Bank accession number is KU507075. Nt LDC1 gene has a 666-bp open reading frame（ORF）, encoding 221 amino acids. The putative protein molecular weight was 24 264.9 Da and its theoretical isoelectric point was 5.43. Sequence alignment displayed that LDC protein from different plants were conserved and that Nt LDC protein showed high similarity to tomato and potato LDC proteins. Evolution analysis result suggested that Nt LDC1 has the nearest genetic relationship with tomato LDC. Real-time PCR result showed that the relative expression level of Nt LDC1 gene in leaf is the highest. Low temperature can induce Nt LDC1 gene expression, the expression level peak at 8 h after low temperature treatment, suggesting Nt LDC1 gene may play a role in the tobacco response to low temperature.