Genska karakterizacija, kloniranje i ekspresija Toll-like receptora 1 mRNA nilske tilapije (Oreochromis niloticus)

Toll-like receptors (TLRs) are the most studied group of pathogen recognition receptor categories that detects infectious agents in vertebrates. Fish TLRs exhibit clear, distinct features, structure and a larger diversity than in other vertebrates. This study focused on identifying and detecting the structure of Oreochromis niloticus (Nile tilapia) Toll- like receptor-1 (TLR1|) as a model in freshwater bony fish. The full-length cDNA sequence of Oreochromis niloticus TLR1 mRNA was cloned. Cloning and sequence analysis revealed that the complete cDNA sequence of Oreochromis niloticus TLR1 consists of 2355 base pairs and encodes a polypeptide of 785 amino acids. The molecular analysis of the amino acid sequence indicated that Oreochromis niloticus TLR1 has the standard structural features and major components of amino acids of TLR family members, and is considered an orthologue to the vertebrate TLR1, not a paralogue. The translated amino acid analysis showed 96%, 88%, 85%, and 85% degrees of identity with Zebra Mbuna, Sea bass, Damsel fish, and Clownfish, respectively; and showed 66% identity t with electric eels and 61% with starlets. Phylogenetic analysis revealed that the Nile tilapia TLR1 is closely related to Larimichthys crocea, Epinephelus coioides, and Takifugu rubripes TLR1. Oreochromis niloticus TLR1 was expressed in the kidneys, brain, spleen, intestines, muscle, liver, gills, heart and skin. Quantitative RT-PCR showed differences in the expression levels between the tested tissues. In conclusion, this study is the first report (according to our knowledge) and provides a complete molecular and functional characterization of Oreochromis niloticus toll-like receptor 1, which is considered to be functionally orthologous to TLR1 in other species models.Toll-like receptori (TLR) najviše su istraživana skupina receptora za prepoznavanje uzročnika bolesti u kralježnjaka. TLR u riba pokazuju jasna razlikovna svojstva, strukturu i veliku raznolikost u odnosu na druge kralježnjake. Ovo je istraživanje usredotočeno na identifikaciju i otkrivanje Toll-like receptora 1 (TLR1) u nilske tilapije (Oreochromis niloticus) kao predstavnika slatkovodnih riba. Klonirana je puna sekvencija cDNA TLR1 mRNA. Utvrđeno je da se kompletna sekvencija cDNA TLR1 nilske tilapije sastoji od 2355 baznih parova i kodira polipeptid od 785 aminokiselina. Molekularna analiza sekvencija aminokiselina upućuje na to da TLR1 nilske tilapije ima standardna strukturna svojstva i glavne komponente porodice TLR receptora i smatra se ortologom, ne paralogom TLR1 kralježnjaka. Analiza prevedenih aminokiselina pokazala je stupanj identičnost od 96 % s mbuna zebrom, 88 % s lubinom, 85 % s damsel ribom i 85 % s ribom klaun, dok je stupanj identičnosti s električnom jeguljom bio 66 %, a s ribom starlet 61 %. Filogenetska analiza pokazala je da je TLR1 nilske tilapije usko povezan s TLR1 vrsta Larimichthys crocea, Epinephelus coioides i Takifugu rubripes. TLR1 nilske tilapije bio je izražen u bubrezima, mozgu, slezeni, crijevima, mišiću, jetri, škrgama, srcu i na koži. Kvantitativni RT-PCR pokazao je razlike u razini ekspresije među testiranim tkivima. Prema našim podacima ovo je istraživanje prvo koje donosi kompletnu molekularnu i funkcionalnu karakterizaciju Toll-like receptora 1 nilske tilapije, te se smatra funkcionalnim ortologom TLR1 u drugih vrsta