Study On The Selection Of Reference Genes And MRNA Levels Of NRAMP Family From Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans (At. ferrooxidans) is an important component in the consortia of micro-organisms used in biomining operations for metal recovery. It is anacidophilic, chemolithoautotrophic, y-proteobacterium capable of aerobic growth via the oxidation of Fe2+ to Fe3+ or reduced inorganic sulfur to sulfuric acid. The range of optimal pH of At. ferrooxidans for growth is from 1.8 to 2.2.The family of NRAMP (natural resistance-associated macrophage protein) metal ion transporters functions in diverse organisms from bacteria to human. Two members of NRAMP family, AFE₂126 and AFE₂920, were found in At. ferrooxidans. Four genes including AFE₂126 of At. ferrooxidans DC, AFE₂920 of At. ferrooxidans DC, AFE₂126 of At. ferrooxidans DY15 and AFE₂920 of At. ferrooxidans DY15 were sequenced. Sequences aligment of gene AFE₂126 revealed that the sequences of At. ferrooxidans DC and standard strain At. ferrooxidans ATCC23270 were 100% indentity, while there were seven nucleotide mutations about AFE₂126 gene between At. ferrooxidans DY15 and standard strain At. ferrooxidans ATCC23270. Howerer, because of the degeneracy of amino acids, there was only one amino acid mutation, which was Thr48 of At. ferrooxidans DC replaced by Met48 of At. ferrooxidans DY15. Sequences aligment of gene AFE 2920 revealed that the sequences of At. ferrooxidans DC and standard strain At. ferrooxidans ATCC23270 were 100% indentity, while there were twelve nucleotide mutations about AFE₂920 gene between At. ferrooxidans DY15 and standard strain At. ferrooxidans ATCC23270. Howerer, because of the degeneracy of amino acids, there was only one amino acid mutation, which was Ala343 of At. ferrooxidans DC replaced by Val343 of At. ferrooxidans DY15.The subcellular localization of four proteins including AFE₂126 of At. ferrooxidans DC, AFE₂920 of At. ferrooxidans DC, AFE₂126 of At. ferrooxidans DY15 and AFE₂920 of At. ferrooxidans DY15 were predicted to lie in the cytoplasmic membrane based on analysis by the PSORTb v2.0 server, and their protein sequences indicated the presence of eleven internal helices based on analysis by TMHMM v2.0 server.After transport motif analysis by Motif Scan server, the results showed that the AFE₂126 protein of At. ferrooxidans DC and At. ferrooxidans DY15 had a conserved transport motif of NRAMP family (5'-QVIASIFMPAAMLFLLMLLNDREIMGSYVNRR-3'), and the AFE₂920 protein of At. ferrooxidans DC and At. ferrooxidans DY15 had a conserved transport motif of NRAMP family (5'-IITAAQSGAQ WGFKLLLLQFVLMPILYIVQELTVRLGIFTGK-3').Seven candidate reference genes from At. ferrooxidans DC were cloned and sequenced, and their TIGR locus and gene symbol were AFE₂121(map,810 bp), AFE₂475 (gmk,639 bp), AFE₂714(rplL, 387 bp), AFE₂150 (gyrA,2538 bp), AFE₂114 (recA,1038 bp), AFE₁680(era,915 bp), AFE₂711(rpoC,4155 bp). Sequences aligment of seven candidate reference genes revealed that the sequences of At. ferrooxidans DC and standard strain At. ferrooxidans ATCC23270 were 100% indentity.We studied the mRNA expression levels of seven candidate reference genes from At. ferrooxidans DC in six different Fe2+ concentration using RT-qPCR (the Reverse Transcription fluorescence-based quantitative real-time PCR), and evaluated their expression stabilities. Descending order of expression stabilities were era, gmk, rpoC, gyrA, recA, map, rplL.We chose the first 3 candidate reference genes(era, gmk, rpoC) as reference genes of this study. The results showed that AFE₂126 mRNA levels in At. ferrooxidans DC were upregulated in response to increases in ambient Fe2+, while mRNA levels of gene AFE₂920 were irregular changes in response to increases in ambient Fe2+...