Desenho de oligonucleotídeos degenerados para isolamento de genes biomarcadores de peixes do gênero astyanax: um primeiro passo visando monitoramento ecotoxicológico no Rio Araguaia

Abstract

The growing human activity on the environment is one of the main factors for the increase in pollution of aquatic environments. In view of this situation, the ecotoxicological monitoring of aquatic environments becomes more essential. Among the tools that can be used for such toxicological analyzes, molecular biology proves to be an excellent tool with the use of RTqPCR (Reverse Transcription Followed by Quantitative Polymerase Chain Reaction) applied to expression analysis as one of the most sensitive techniques in the evaluation of the exposure of bioindicator organisms to pollution. Thus, this study aimed to develop degenerate oligonucleotides for the genus Astyanax, which can be used in future studies for toxicological evaluations using this genus as a bioindicator. Considering this, nine genes with biomarker potential already described in the literature were then selected, aiming at the development of degenerate oligonucleotides for use in future research on ecotoxicological monitoring, in aquatic environments on the Araguaia River. The nine selected genes, acetylcholinesterase (ACHE), metallothionein (MT), cytochrome P450 family 1 A (CYP1A), catalase (CAT), superoxide dismutase (SOD), immunoglobulin M (IgM),cyclooxygenase 2 (COX2), interleukin 1 beta (IL-1B) and beta chain T cell receptor (TCRb) were chosen from a bibliographic review prioritizing the most commonly used genes, as well as the responses of these genes to pollutant exposure and oxidative stress. The oligonucleotides (primers) were then generated using two different methodologies. In the first, the amino acid sequences for each protein of the selected genes were aligned and the most conserved regions at the amino and carboxy-terminal ends of the alignment were selected. Later, the alignment of the nucleotide sequences was performed and the regions selected in the previous alignment were marked. The oligonucleotides were then designed and from these regions, replacing the degenerated bases with the respective IUPAC codes. In the second methodology, the pair of forward and reverse oligonucleotides was generated using the jCODEHOP software that design the pair of primers from the alignment of the amino acid sequences. The oligonucleotides generated by both methods had their degenerations, melting temperatures (Tm) and CG content (Cytosine and Guanine) calculated. It was observed that the oligonucleotides generated by jCODEHOP might be more adequate, since they presented less degeneration and less GC content, which can reduce the chances of unspecificity in the PCR reaction. The degenerate primers designed at this work are proposed as a tool for future molecular analyzes applied to ecotoxicology of aquatic environments using species of the genus Astyanax as a model.