Larval Developmental Expression Of Pepsinogens, Cloning And Expression Of Two Gastrin Family Genes In The Mandarin Fish (Siniperca Chuatsi)

Pepsinogen is the precursor of pepsin, which can be activated in thecondition of gastric acid. The full length cDNAs of three kinds ofpepsinogen genes, PG A1, PG A2and PG C were obtained in our previouswork. To further understand the the expression patterns of these pepsinogengenes, pepsinogens mRNA expression levels during early developmentalstage (0-22(days post hatching, dph) were analyzed by real time PCR. Theresult showed expression of three pepsinogen genes started from8dph,then in a rising tendency. PG A1and PG A2had similar expression levelsduring0-19dph, PG C was the lowest. During16-22dph, the expression ofPG A2was stable. Pepsinogen gene levels emerged before13dph wereprobably expressed by other tissues before gastric gland, otherwise thegastric gland cells may emerged around the start point of expression ofthree pepsinogens.Pepsin, the activated pattern of pepsinogen, is an important digest enzyme in the digestive tract and the executor of acid digestion. It take animportant role in primary digestion of proteins in food. The measure ofactivity of pepsin reflects digestive capacity of mandarin fish’s stomach.During the larval developmental stage, the stomach has not developmented,digestion of food mainly depends on intracellular digestion and alkalineproteolitic enzymes, which is then replaced by pepsinogen in the digestivetract. It exhibit a complex change during larval development of mandarinfish. A low level of pepsin-like activity was detected during0-8dph beforeemerging of gastric gland and expression of pepsinogen genes. This maycame from maternal effect or other aspartic proteinases. The activityundergo a rapid rising after8dph which is consistent with the starting ofpepsinogen mRNA expression.The secretion of pepsinogen influenced by many factors. It is knownthat gastrin and cholecystokinin can regulate the secretion of pepsinogen.They are members of gastrin family, gastrin could stimulate acid secretionin stomach, cholecystokinin conduct gallbladder emptying and pancreaticsecretion. Few work has been done about the function of gastrin andcholecystokinin in fish. Two intestinal hormone genes, gastrin (GAS) andcholecystokinin (CCK), were cloned from mandarin fish (Sinipercachuatsi). The GAS cDNA consists of581bp nucleotides, including a100bp5’ untranslated region (UTR), a336bp open reading frame(ORF) encoding111amino-acid residues, and a145bp3’ UTR. The CCK genehas two isoforms, CCK1and CCK2, consisting of843bp and846bpnucleotides, respectively. The CCK1gene consists of a60bp5’untranslated region (UTR), a414bp ORF encoding a polypeptide of137amino-acid residues and a369bp3’ UTR, while the CCK2gene consists ofa112bp5’ untranslated region(UTR), a403bp ORF encoding apolypeptide of134amino-acid residues and a332bp3’ UTR. TheC-terminal of GAS polypeptide has a similar octapeptide structure(DYQGWVDF/DYLGWMDF) to that of CCKs’. The only differencesoccur in the third and the sixth amino acid in the C-terminal, Val3(GAS)and Glu6(GAS) are replaced by Met3(CCK) and Leu6(CCK), respectively.Real-time PCR analysis revealed that the GAS mRNA was mainlyexpressed in the intestinal and pyloric caecum. The highest CCK1andCCK2mRNA expression was detected in the brain, and also detected inintestinal and pyloric caecum. These evidences suggest that GAS andCCKs may be involved in regulating the digestion process, while CCKsmay be associated with the digestion and neurosecretion process. The GASand CCK genes are constitutively expressed during the larval developmentstage (0-22days post hatching, DPH). The expression at early stage (4-8DPH for GAS and2-4DPH for CCK) was higher, which may be related todevelopment and growth of the digestive tract, and then gradually reduced to a stable level at late stage.