Lead (Pb) is one of the highly persistent, toxic, and widely distributed heavy metal pollutants in the environment. This heavy metal has a tendency to enter human food chain, thus affecting public health. One effective way to remove heavy metals pollutant is by using plants, a technology known as phytoremediation. One such plant that is routinely employed as an experimental model for such studies is Nicotiana tabacum. As tobacco plants are not generally consumed by herbivores, it minimizes the possibility of Pb from entering food chain.
A number of studies suggest that annexins, a calcium-binding protein, does play a role in plant stress response. The expression of annexin gene in plants appeared to be regulated by tissue-specific developmental and environmental signal. A vacuole-associated annexin from N. tabacum, Anx3, was investigated, to observe the involvement of this gene in Pb-induced stress.
Reverse transcription following quantitative real-time 0olymerase chain reaction (qRT-PCR) is a useful analysis to study gene expression. In this analysis, a reference gene that acts as internal control (housekeeping gene) is routinely employed for normalization of qRT-PCR against the target gene (Anx3). The candidate reference genes, L25, EF-1α, and Ntubc2, were evaluated using suitable primer pairs in order to select the most stable reference gene for normalization of qRT-PCR in this study.
Using geNorm, NormFinder, and BestKeeper programs, the most suitable reference gene identified in this study was L25. The relative quantification of Anx3 gene expression normalizing against L25 was accomplished by REST software. The expression level of Anx3 in Pb-treated N. tabacum was upregulated by 2.2-fold (p < 0.05). The experimental methods used and the participation of Anx3 in defense against Pb stress will be discussed
