Identification, Tissue Distribution And Feeding Related Changes Of SPX And CB1 In Schizothorax Prenanti

In order to study CB1 and SPX on their regulation appetite in Schizothorax prenanti, we mainly used RACE and quantitative PCR (qPCR) techniques. In this study, we have examined that the expression of CB1 and SPX mRNA in different organizations and the effect of nutrient levels (periprandial, fasting and refeeding) on the expression.Our results showed that we obtained CB1 and SPX Sequence, with 1234bp and 1330 bp respectively, and tested mRNA levels in the forebrain related to periprandial, fasting and refeeding in S. prenanti.According to the results, CB1 and SPX are widely distributed in the organization, and express the amount the highest in the forebrain. Sequence analysis found that the CB1 and SPX of S. prenanti are highly conserved in fish. Structure prediction showed that transmembrane region of S.prenanti CB1 is similar with zebrafish on the sequence location. SPX contained a 31 amino acid signal peptide region, and a 14 amino acid mature peptide was sheared by RR and GRR.Among them, the CB1 also expressed in the pituitary gland, heart, skin is relatively abundant, and expressed in the gonads minimum; SPX in pituitary, foregut, midgut tissue expression is relatively high, and almost no expression in the muscles.Using real-time RT-PCR to examine 7 groups periprandial (pre- and post-feeding) changes of CB1 and SPX mRNA expression at-3 h,1 h,0 h,+1 h and+3h 5 time points in S. prenanti forebrain. Our data showed that CB1 mRNA of the unfed groups at +3 h post-feeding point appeared significantly higher than fed groups (P<0.01). Compared with unfed group, the fed group of CB1 mRNA in +1 and+3 decreased significantly (P< 0.01); On the contrary, the Ya-fish SPX mRNA expression of unfed fish significantly decreased in the forebrain at 1 and 3h post-feeding compared to preprandial groups, respectively (P<0.01). Compared with fasting group, SPX expression significantly increased after a meal. Fasting test lasted for 9 days and there were 9 groups in total which were 5 groups for fasting and the remaining for free food intake. Among the fasting groups,2 groups were refeeding on the 7th-day. Tissues were collected on 3th-,5th-,7th- and 9th-day respectively. These tissues were used for testing the expresion of SPX in S. prenanti forebrain. The rerults showed that CB1 of unfed groups were increased significantly on 3th-,5th-, and 7th-day in S. prenanti forebrain (P<0.01). On the contrary, SPX mRNA expression of unfed fish decrease compared to 3th-,5th- and 7th-day fed animals (P< 0.01). When 7th-day, another food-deprived group were refed, the CB1 mRNA expression back to normal level, but SPX mRNA expression significantly increased than 3th-,5th- and 7th-day fasted fish and 1.2-fold higher than control group (P<0.01). After the initiation of refeeding, at 9th day, the expression of SPX mRNA downgrade to control levels.Conclusion:1、The partial CB1 and full-length SPX gene sequence were first cloned from the brain of S. prenanti and showed high conservation in fish.2、CB1 and SPX is widely distributed in the tissue organ of S. prenanti, and highest expression in the forebrain.3、CB1 and SPX mRNA changed with the energy state, and related to feeding regulation. Food deprivation led to CB1 expression increased in S. prenanti, while SPX expression decreased.