Molecular Cloning And MRNA Expression Of Ghrelin In Southern Catfish (Silurus Meridionalis)

Ghrelin is identified as the first endogenous ligand for the growth hormone(GH) secretagogue receptor(GHSR).A amino acid peptide with a fatty acid chain modification on the N-terminal third amino acid, which was originally extracted from the rat stomach by kojima and also widely expressed in different tissues. The discovery of Ghrelin has broadened our understanding of the regulation of energy homeostasis in vertebrates. Studies in teleosts reveal that Ghrelin is expressed at sites beyond the stomach including brain, intestine, pancreas, gall bladder, kidney and gill.It was shown that Ghrelin was related to a series of biological functions and effects including stimulate GH release, promote food intake, adiposity. Also influence mang other hormone release and regulate GH/IGFF-1axis.Those functions which through bingding to growth hormone(GH) secretagogue receptor-1a(GHSR-1a).The full-length473bp cDNA encoding for prepro-ghrelin was cloned from China southern catfish using RT-PCR and rapid-amplification of cDNAends (RACE). The southern catfish prepro-ghrelin cDNA was473bp in length, and consisted of a45bp5’untranslated region, a104bp3’-untranslated region and324bp open reading frame (ORF). The predicted mature ghrelin peptide contains22-amino acid. The N-terminus is more conserved than its C-terminal portion. At the N-terminus of the mature ghrelin peptide, there is a strong conservation of the first7AA (GSSFLSP), Its biological activity is decided by the N-terminal sequence, and the first4AA "GSSF" are considered to be the "’biological active core" of the ghrelin peptide. The amino acid sequences of mature ghrelin in southern catfish showed45%-81%homology with teleost and32%-46%with mammals, anians and amphibians. The "biological active core" of the ghrelin peptide in mammals, amphibians, anians and teleost were well conserved through evolution. It suggests that the biological activity may inherit stability, so ghrelin may plays an important role in physiological process. The expression of prepro-ghrelin in tissues distribution was quantified by real-time PCR. A strong signal was observed in stomach and gut, and it is similar to the results studied before. This widespread distribution of the ghrelin suggested this peptide may involve regulating multiple biological functions in fish. During embryonic development stages, the expression signals of ghrelin appeared very weak in all stages, but the stage before first-feeding larva is significantly higher to other stage; especially all egg yolk has been almost consumed. It explained that after first-feeding stage, each organs of southern catfish made important coping mechanism to adapt the food conversion from endogenous to exogenous, and gerelin made an important role in regulating endogenous food, however, it had limited role in regulating exogenous food.We detected the prepro-ghrelin mRNA expressions as food intake changed, the results showed that expressions of prepro-ghrelin mRNA levels were significantly higher (p<0.05) at feeding time (0h) than2h before and after feeding time. A lower prepro-ghrelin mRNA levels at2h may due to satiation and a higher prepro-ghrelin mRNA levels at Oh suggested ghrelin might be a hungry signal participating the regulation of food intake in southern catfish. Besides, the levels reduced at2h indicated that ghrelin did not participate the food digest process, just as an appetite provoke signal. The expression of prepro-ghrelin mRNA levels increased with fasting day elongated and the highest levels (p<0.05) appeared at fasting for2days, but when the fish were continued fasted for7days and14days, the expression of prepro-ghrelin mRNA levels reduced, In the refeeding experiment, the expression of prepro-ghrelin mRNA levels of4hours after refeeding reduced rapidly compared to fasting for14days, Meanwhile, we also found prepro-ghrelin mRNA levels of24hours after refeeding increased compared to4hours after refeeding, and returned about50%compared to fasting1day. From that we conjecture that ghrelin regulating food intake may belong to a short-term mechanism.In conclusion, using molecular cloning techniques, a full-length473bp cDNA encoding for prepro-ghrelin was cloned from China southern catfish. The predicted mature ghrelin peptide contains22-amino acid. The first4AA "GSSF" are considered to be the "biological active core" of the ghrelin peptide. The expression levels of ghrelin mRNA in the stomach was measured higher. Meanwhile ghrelin may regulate at the early stages of life. And ghrelin did not participate the food digest process, just as an appetite provoke signal. We conjecture that ghrelin regulating food intake may belong to a short-term mechanism.