| Orexin and CART (cocaine-and amphetamine-regulated transcript) which belong to hypothalamic neuropeptides, are important feeding regulatory factors. Orexin is an orexigenic factor, which may stimulate appetite and increase food intake, however, CART is an anorexigenic factor, which may inhibit appetite and decrease food intake.The research collected southern catfish as research subjects, the full-length cDNA of orexin and CART were cloned using RT-PCR and RACE, and functions of the encoding amino acids were analyzed using bio informatics software. Orexin mRNA and CART mRNA expression level were studied by qPCR in brain, liver, kidney, heart, stomach, gut and gills, in early developmental stages including oosperm stage, blastoeyst stage, gastrula stage, neurula stage and before first-feedingand stage, and in different time points of juvenile brain tissue.The results were as follows:1.Orexin cDNA contained707nucleotides with an open reading frame of474nucleotides encoding a158-amino acid peptide, which contained a38-amino acid signal peptide, a48-amino acid orexin-A, a28-amino acid orexin-B, a38-amino acid Carboxy-terminal and two proteolytic site.CART cDNA contained688nucleotides, with an open reading frame with357nucleotides encoding a118-amino acid peptide, which contained a25-amino acid signal peptide.2. Molecular weight, isoelectric point and average hydrophilic of orexin were17.45KD,9.67and-0.382. The protein consisted of19amino acids,and the content of leucine and phenylalanine was about15.80%and0.60%, respectively. Molecular weight, isoelectric point and average hydrophilic of CART were13.33KD,8.18and-0.147.The protein consisted of20amino acids, and the content of leucine and histidine, tryptphan and tyronsine were about14.41%and0.85%,respectively. Secondary structures of orexin and CART were composed of alpha helix, beta folding, random coil and extend chain.3.Orexin-A of southern catfish had56.3%,50.0%,47.9%,45.80%,33.3%and37.5%similarity with orexin-A protein from zebrafish, goldfish, grouper, winter flounder, Atlantic cod and human respectively. Orexin-B of southern catfish had75.0%,75.0%,60.7%,64.3%,55.2%and50.0%similarity with orexin-B protein from zebrafish, goldfish, grouper, winter flounder, Atlantic cod and human respectively. Phylogenetic tree displayed that, Orexin of southern catfish was clustered closely with goldfish and zebrafish, and CART of southern catfish was clustered closely with channel catfish. The results showed that, CART of southern catfish had95.8%,72.0%,75.0%,72.9%,75.0%,72.0%,61.9%,69.5%,48.3%and52.5%similarity with CART from channel catfish,goldfishl, goldfish2, common carp1,common carp2, Atlantic cod, winter flounder, Atlantic salmon, winter skate and human respectively.4. The mRNA expression of orexin and CART in brain was significantly higher than the other kinds of peripheral tissues. The mRNA expression of orexin had no striking difference among peripheral tissues.5.The mRNA of orexin and CART expressed in all stages except in oosperm, and the orexin mRNA and CART mRNA expression showed an upward trend with the development.6. Under the conditions of normal feeding, the mRNA expression level of orexin at0h was significantly higher than the other periods, and the mRNA expression of CART was lower than after mealtime levels.Under the conditions of starvation,Orexin mRNA levels was increased at starvated48h, however orexin mRNA and CARTmRNA levels were decreased at1week and2weeks.The conclusions suggested in this study were as follows:1.Phylogenetic tree showed that, orexin of southern catfish was clustered closely with goldfish and zebrafish, and CART was clustered closely with channel catfish.2.The mRNA of orexin and CART were mainly expressed in the brain, which is in accordance with their functions of regulating food intake.Comparing with existing research,it is found that the mRNA expression of orexin and CART in peripheral tissues is different among species.3.Orexin mRNA and CART mRNA were detected in the earlier period of embryonic development, and gradually increased along with the development. Nervous system and organs formed in the neurula stage and organ differentiation stage, which showed that the gene expression was earlier than the formation of the nervous system. The yolk was the main energy source of the embryonic stage.With the development of the yolk depleted, the body needs food intake from the outside, meanwhile,to prevent the outside shortage of food and the presence of toxic substances in the food,as a result orexin mRNA and CARTmRNA expression were increased. 4. The food in the stomach would be depleted gradually at0h, thus the mRNA expression level of orexin was increased, but CART mRNA reduced. The stomach was filled after feeding time and the body had a strong satiety. The mRNA expression level of orexin was reduced, but CART mRNA was increased. Orexin mRNA expression were increased during short-term hunger so that the body remain to be feeding state, in order to get much more food to provide energy. This was an active regulation. During chronic hunger, in order to resist the adverse external environment, the body reduces the function of the various systems, hence the orexin mRNA and CART mRNA was lower, it was a passive adjustment. Therefore, we speculated that they maybe involved in food intake in southern catfish. |
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