Cloning And Expression Analysis Of IGFBPs Gene In Japanese Flounder

Insulin-like growth factor-binding proteins (IGFBPs) play a pivotal role inregulation system by regulating the biological activities of IGFs as well as theirsIGF-independent functions. In this study, we cloned and characterized cDNA sequencesof six insulin-like growth factor binding proteins (IGFBP-2a,-2b,-3,-4,-5,-6) fromJapanese flounder, Paralichthysolivaceus. The full-length cDNA of IGFBP-2a is1046long and consisting an open frame (ORF) of876bp, a5’-untranslated region (UTR) of125bp and a3’-UTR of45pb. IGFBP-2b is1067bp, including a5’-UTR of53bp, a3’-UTR of198bp, and an ORF of816bp. IGFBP-3was1070bp long and consisted a825bp long open-reading frame (ORF) encoding a predicted polypeptide of283aminoacid residues. The complete cDNA sequence of IGFBP-4was1096bp in length with a780bp ORF coding for a259amino acid polypeptide. IGFBP-5was1297bp consistingORF that encoding a267amino acid polypeptide. The partial sequence of IGFBP-6including a complete ORF of603bp coding a201amino acid polypeptide. The overallstructure is conserved and related with highly degree to each other. But each IGFBP hasunique properties, and performs the functions of potentiating or inhibition of thebiological activities of IGFs. Real-time quantitative PCR results revealed that fourIGFBPs mRNA were rifely expressed in all detected tissues, but the expression patternwere different among each other. Interestingly, IGFBP-2a,-3,-4,-5,-6mRNAexpression levels in almost all detected tissues were higher in female than male, butof IGFBP-2b was precisely the opposite. At different embryonic and larval developmentstages, IGFBP-2a,-2b,-3,-4,-5and-6mRNAs were widely observed in all examined stages, nevertheless, different IGFBPs vary a lot in expression patterns. InSodium salt (T4, the main form of Thyroid hormone in animals)-treated and thiourea(TU)-treated larvae, T4and TU affect the expression of six proteins in varying degrees.The expressions of IGFBP-2a had no visibly change, except in T4-traeted17dph larvae.These results provided basic information for further studies on the role of IGF system inflatfish development and metamorphosis.For further study on the relationship between Thyroid hormone (TH) and IGFBPs,we treated the Japanese flounder embryonic cell with different concentration gradienttriiodothyronine (T3) and thyroxine (T4), sampled at different time points, and thendetected the expression levels of seven IGFBPs by Real-time qPCR. All the IGFBPsexpression levels were affected by two kinds of TH, the effect of T3was faster than T4,but the effect of T4was more apparent than T3. This result further proved that T4playsthe main role in metamorphosis of Japanese flounder; T4would be transformed into T3form in the process of working on metamorphosis, so the effect of T3was faster.This study provided basic data and information for the further studies on therelationship between IGFs system and metamorphosis in flatfish.