Studies On Gene Clone And Expression Regulation Of Insulin-like Growth Factors And Binding Proteins In Japanese Seabass (Lateolabrax Japonicus)

Japanese seabass(Lateolabrax japonicus) is considered as one of the mosteconomic marine fishes and widely cultured along China Coast. This study tookJapanese seabass which cultured in Qingdao city, Shandong province as anexperimental fish, and molecular biology methods was applied to study the gene cloneand expression regulation of Insulin-like growth factors(IGF) and their bindingproteins. This study fills up the blank in the study of IGF family in Japanese seabass,and lays a foundation for the future study on the regulation of IGFs expression andtheir physiological function.1The gene clone and expression analysis of Insulin-like growthfactor-1,2(IGF-1,2) in Japanese seabass (Lateolabrax japonicus)The technique of RT-PCR, Rapid Amplification of cDNA ends(RACE)are usedto obtain a complete cDNA sequence of Japanese seabass (Lateolabraxjaponicus)IGF-1and IGF-2. The full-length of IGF-1is876bp, encoding185aminoacid and the full-length of IGF-2is798bp, encoding215amino acid. The sequenceanalysis is conducted by the biological software and authoritative website, and theresults shows that both ligands are well-conserved in the evolutionary status, both ofthe deduced proteins are subdivided into six structural domains, including a signalpeptide, B, C, A, D domains and E peptide. The two ligands both contain theconserved functional structures which involves in the combination with the ir receptorsand binding proteins. The technique of florescent real-time quantitative RT-PCR wasapplied on the study of IGF-1and IGF-2mRNA expression pattern in differenttissues.The result of IGF-1mRNA expression in different tissues shows that itshighest expression was in liver, relative high expression was inintestine, cecum,muscle, spleen, other tissues have the less expression of IGF-1.The expression pattern of IGF-2is relatively wide, almost every detected tissue transcripts IGF-2, its highestexpression is in heart, brain, gill, stomach, cecum, intestine, kidney, gonad, spleen andmuscle has the relatively high expression.2The gene clone and expression analysis of Insulin-like growthfactor-binding protein-1,2(IGFBP-1,2) in Japanese seabass(Lateolabrax japonicus)The technique of RT-PCR, Rapid Amplification of cDNA ends(RACE) are usedto obtain a complete cDNA sequence of Japanese seabass (Lateolabraxjaponicus)IGFBP-1and IGFBP-2. The full-length of IGFBP-1is1779bp, encoding248amino acid and the full-length of IGFBP-2is815bp, encoding267amino acid.The results of multiple amino acid analysis of IGFBP-1and IGFBP-2in Japaneseseabass (Lateolabrax japonicus) shows that both proteins contain a C-terminal,N-terminal and a variable central L-domain as other teleost’,18conserved cysteineresidues also could be found which are considered to contribute to the stability ofamino acid structure. Both proteins contain the CGCCXXC-box in the N-terminal andthe CWCV-box in C-terminal, these two special structures are considered to beinvolved in the combination with their related ligands. Besides, there is a structurenamed as RGD motif in the C-terminal of IGFBP-2. This motif was shown to interactwith integrin, a family of cell adhesion receptors that is involved in cell-to-cellinteraction and intracellular matrix association. The technique of florescent real-timequantitative RT-PCR was applied on the study of IGFBP-1and IGFBP-2mRNAexpression pattern in different tissues. The result shows that the highest expression ofIGFBP-1mRNA is in liver, the higher expressions are found in muscle and spleen.The highest expression of IGFBP-2mRNA is in liver too, and muscle has lessexpression of IGFBP-2mRNA, other tissues express much less.3Study on the effect of exogenous hormone injection on the serumIGF-1and IGF-1mRNA and IGFBP-1mRNA expression in theliver of Japanese seabass (Lateolabrax japonicus) This study focus on the effect of luteinizing hormone releasing hormone-A(LHRH-A) and human chorionic gonadotropin(HCG) on the IGF family genes ofJapanese seabass(Lateolabrax japonicus). Intraperitoneal injection was applied, andthe serum IGF-1level was examined by Radiate Immuno Assay (RIA) after6h,12h,24h and48h. Besides, the technique of florescent real-time quantitative RT-PCR wasapplied to examine the changes of expression level of IGF-1mRNA and IGFBP-1mRNA in liver at the same time. There are three groups defined as PS (PhysiologicalSodium) group, LHRH-A group and HCG group. The result shows that, the serumIGF-1level in LHRH-A group doesn’t change much during the first12h, but after24hit begins to decrease and stay at a relatively low level. The expression level of IGF-1mRNA increase significantly compared to PS group, but it decreases to the level equalto PS group and stay unchanged when the experiment is over. The expression level ofIGFBP-1mRNA decreases at the whole time but the difference is not significant to PSgroup. In the HCG group, the serum IGF-1level decreases significantly at12h, after24h, it continues to decrease and stay at a relatively low level at the whole time. Theexpression level of IGF-1mRNA doesn’t change at24h and has no difference with PSgroup but at48h, it decreases significantly. The expression level of IGFBP-1mRNAincreases significantly at12h, but after24h, it starts to decrease and the level stayhigher than the PS group at the rest of the experimental time. This study suggests that,both LHRH-A and HCG could trigger the growth process of Japanese seabass(Lateolabrax japonicus), but the specific mechanism is still unknown, but the positiveeffect of LHRH-A on growth may result from the influence of GH produced bypituitary, which means LHRH-A indirectly regulates IGF family genes throughstimulating pituitary producing GH. The mechanism may be different from the onewith LHRH-A.