NtNramp1基因参与不同镉积累基因型烟草品种镉积累差异的功能解析

Functional analysis of Nt Nramp1 pariticipating in Cd accumulation in tobacco of different Cd accumulating genotypes

【目的】探究不同品种烟草镉积累差异的分子机制。【方法】采用同源克隆方法从烟草不同镉积累基因型品种中克隆拟南芥At Nramp1同源基因Nt Nramp1;应用生物信息学方法分析烟草不同镉积累基因型品种中Nt Nramp1基因序列差异;通过酵母异源表达系统研究烟草不同镉积累基因型品种的Nt Nramp1对镉的转运活性差异。【结果】金英和Komotini Basma两个烟草品种对镉的积累存在显著差异;从镉积累显著差异的两个烟草品种中克隆到的Nt Nramp1基因(分别命名为Nt Nramp1a和Nt Nramp1b)编码区存在57个单碱基差异,其中Nt Nramp1a 1305位碱基变异(G>A)导致所编码蛋白比Nt Nramp1b编码蛋白缺少C端两次跨膜;酵母异源表达结果表明来自低镉含量品种金英的Nt Nramp1a转运镉的活性比来自高镉含量品种Komotini Basma的Nt Nramp1b转运镉活性显著降低。【结论】金英和Komotini Basma两个烟草品种中Nt Nramp1蛋白活性差异是导致两烟草品种镉积累差异的重要原因。

The present study was conducted to investigate the molecule mechanism of different cadmium accumulation ability among tobacco varieties. Based on At Nramp1 sequence of Arabidopsis thaliana, Nt Nramp1 gene from Jinying and Komotini Basma with different cadmium accumulation ability were cloned and their sequence was analyzed by bioinformatics methods. Yeast heterologous expression system was used to analyze different cadmium transport activity of Nt Nramp1. Results showed that Jinying and Komotini Basma were two tobacco varieties(Nicotiana tabacum L.) that had significant difference of cadmium accumulation ability. Nt Nramp1 cloned from Jinying and Komotini Basma were named as Nt Nramp1 a and Nt Nramp1 b respectively. There were 57 single nucleotide polymorphisms(SNP) in the coding region between Nt Nramp1 a and Nt Nramp1 b. The SNP in 1305 bp of Nt Nramp1 a led to premature termination of protein translation, which resulted in missing of two transmembrane structure in the C-terminal compared with Nt Nramp1 b. Heterologous expression of Nt Nramp1 a and Nt Nramp1 b in yeast showed that the yeast strain expressing Nt Nramp1 b was more sensitive to cadmium than the that expressing Nt Nramp1 a, suggesting that cadmium transport activity of Nt Nramp1 b was significantly stronger than that of Nt Nramp1 a.Different cadmium transport activities of Nt Nramp1 between Jinying and Komotini Basma resulted in significant cadmium accumulation difference between the two tobacco varieties.