| Large yellow croaker (Pseudosiciaena crocea, Perciformes, Sciaenidae) is a famouseconomic native species in China Seas. Delicious, tender and nutritious, it has become akind of shopping-rush goods in China. The commercial value is high, as well as the scale offarming.During farming, the puberty of lage yellow croaker came earlier during the lastyears. This situation caused a large pecuniary loss and broke the confidence of the public tothis farming fish. To realize healthy aquaculture, a research and study of the puberty oflarge yellow croaker has passed the point of no return.Mainly controlled by the hypothalamus-pituitary-gonad axis, puberty is regulatedby a lot of factors, one of them is gastrointestinal peptides or gut peptides. Gastrointestinalpeptides are released by gastrointestinal tract. They are one of the major regulatory factorsabout ingestion and energy balance. But their physiological action are far beyond these.They have effect on release of hormones, gastro-intestinal fluid delivering, immune system,hematopoietic function, shock inhibition, liver diseases, etc. They also can affect the re-productive system and many of them are confirmed to advance puberty, especially on therelease of GnRH and kisspeptin, two major homones regulating puberty. But the effect ofthem to the large yellow croaker is still unclear. Cutting-edge technologies such as reversetranscription PCR, RACE, fluorescent quantitation real-time PCR and related softwareanalysis was uesed, and three important gastrointestinal peptides were studied. The overalllength sequences were cloned and the expression characterizations were analyzed.By homologous sequence downloaded from genbank, primer were designed and re-verse transcription were used to obtain partial sequences. Then RACE was used to acquirethe full length of cholecystokinin, ghrelin and leptin cDNAs. By then the expression of themRNA in different tissues ware examined by fluorescent quantitation real-time PCR(FQRT-PCR), as well as their response to fasting. The paper’s main results were as follows:Cholecystokinin cDNA (GenBank Accession No.: KC899121) was900bp in length,and a166bp5′-untranslated sequence, an open reading frame consisting of167to574bp, astop codon of TAA, a326bp3′-untranslated sequence, and a poly (A) tail of21bp wereincluded. Cholecystokinin consisted of137amino acids with a calculated molecular massof14.7kDa. High similarity with other cholecystokinin from Perciformes and low similaritywith nonhomologous species were shared. Cholecystokinin consisted of a domain ofYVGWMDF which was highly conservative in all species. The mRNA transcripts of cho-lecystokinin could be detected widely in all the examined tissues with remarkable differentexpression levels. The maximum expression level in the brain tissue and the stomach, and the minimum expression level in the muscle and the splenic organ were showed. Duringfasting, cholecystokinin level in the brain tissues were decreasing while rising in the smallintestine.Ghrelin cDNA (GenBank Accession No.: KC899122) was697bp in length, and a97bp5′-untranslated sequence, an open reading frame consisting of98to424bp, a stop codonof TGA, a273bp3′-untranslated sequence, and a poly (A) tail of20bp were included.Ghrelin consisted of108amino acids with a calculated molecular mass of12.1kDa. Highsimilarity with other ghrelin from Perciformes and low similarity with nonhomologousspecies were shared. Ghrelin consisted of a domain of GSSFL which was also highly con-servative in all species. The mRNA transcripts of ghrelin could also be detected widely inall the examined tissues with remarkable different expression levels.The maximum expres-sion level in the stomach, and the minimum expression level in the adipose tissue and thesplenic organ were showed. During fasting, the level of ghrelin expression showed a ten-dency to rise wavely.Leptin cDNA (GenBank Accession No.: KC899123) was1290bp in length, and a142bp5′-untranslated sequence, an open reading frame consisting of143to628bp, a stop co-don of TGA, a662bp3′-untranslated sequence, and a poly (A) tail of24bp were included.Cholecystokinin consisted of161amino acids with a calculated molecular mass of17.8kDa.High similarity with other leptin from Perciformes and low similarity with nonhomologousspeices were showed. Leptin was characterized by four conserved α-helix domains. ThemRNA transcripts of leptin could be detected widely in all the examined tissues with re-markable different expression levels. The maximum expression level in the liver wasshowed and there were no significant differences among the expression levels of other tis-sues. During fasting, the level of leptin expression showed a tendency to fall by degrees.This paper can be a theoretical base of the study of the puberty of lage yellow croakerand help to sovle the problem of pubertas praecox of large yellow croaker. |
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