Expressão gênica diferencial em tambaquis, colossoma macropomum (cuvier, 1818), expostos ao petróleo e a hipoxia

Abstract

Petroleum industry is a reality in the Amazon region. Specific sediment and pH, along with water level changes, which induce specific patterns of oxygenation and floating vegetation, make such a complex environment that becomes difficult to predict all impacts of an oil spill in the Amazon basin. The present work investigates the effects of petroleum exposure on Tambaqui, an endemic Amazon fish species, Differential Display RT-PCR was applied to reveal differential expressed genes between juveniles exposed and non-exposed to petroleum; sub-letal concentrations, twice letal concentration at three and six hours. Recovery was measured after 6 hours under both conditions: hypoxia and normoxia. After the experiments fish were killed and liver was immediately excised and frozen in liquid nitrogen. RNA was then extracted. cDNA was obtained after hybridization with poli-T primers anchored with terminal nucleotides C, G, or A. Amplifying reaction was performed combining poli-T primers with five heterologous primers with 13 nucleotides. Amplified product was visualized after electrophoresis in 2.5% agarose gel and stained with ethidium bromide. The differentially expressed bands were eluted and cloned with E.coli Vector P-gen easy system, and sequenced. The sequences were compared with EST s public banks. Four genes were identified: (i) RAG2 (recombination-activating genes); (2) ERK (mitogen-activated protein kinase); (iii) GR (glutathione reductase); e (iv) CFTR. The last three are well reported as detoxifying agents while the first regulates cell formation for immune system. RAG2 and ERK are also involved in cell apoptosis. GR is involved in the antioxidant process after environmental stress exposure. CFTR regulates Calcium uptake at fish branchial level. Thus, DD RT-PCR is an efficient method to identify the main biochemical pathways involved in the responses of fish to petroleum exposure.