Identification And Nutritional Regulation Of Leptin Receptor In Grass Carp(Ctenopharyngodon Idella)

Leptin receptor(LEPR)is a type of transmembrane receptor and a member of the class I cytokine receptor family.LEPR plays a decisive role in the physiological functions mediated.In this study,the LEPR of grass carp was selected as the research object,and its c DNA sequence was cloned by RT-PCR,and its space-time expression pattern was analyzed by q RT-PCR.The regulation of Leptin(Lep)and Soybean Isoflavone(SIF)on the expression of grass carp was investigated by using in vitro intestinal cell model.The effects of different nutritional dipeptide Ala-Gln levels,different protein levels and different protein sources on intestinal LEPR of grass carp were investigated by nutrient growth experiment.The main results of this experiment are as follows:(1)Molecular identification and spatiotemporal expression characteristics of LEPR from C.idella.The open reading frame(ORF)sequence of the Ci LEPR gene included 3,504 bp,which encoded 1078 amino acids and the molecular weight of the protein was 121.2KDa and the isoelectric point was 5.06.Sequence analysis revealed that Ci LEPR formed six functional domains,including five FN3 domains and a lep＿receptor＿Ig domain.The five FN3 domains were located at amino acid residues 180-261,363-458,475-557,580-661,and 675-762 and the lep＿receptor＿Ig domain was located at amino acid sites 273-351.The phylogenetic analysis showed that the Ci LEPR amino acid sequence was closely related to that of LEPR from Hypophthalmichthys molitrix,followed by LEPR from Cyprinuss carpio.LEPR m RNA expressions were performed using q RT-PCR in different tissues from C.idellus.The results revealed that Ci LEPR was broadly expressed in the intestine,liver,kidney,spleen,brain,blood and gill,with the highest expression levels in the gill.There was no significant difference in the amount of expression in the spleen and blood.In addition,we analysed the effect of day and night on Ci LEPR expression.The results revealed that the Ci LEPR m RNA levels showed diurnal variation in the intestine.The highest expression was observed at 3:00 and 24:00,and the lowest was observed at 9:00.(2)Effects of recombinant leptin and SIF on intestinal Lep,LEPR expression in C.idella.The cDNA open reading frame(ORF)sequence of Lep gene was cloned from grass carp and the recombinant plasmid p GEX-4T-1-lep was constructed and expressed in Escherichia coli,further optimizing the expression conditions of Lep in grass carp and producing recombinant Lep with high biological activity.It was found in the intestinal cell experiment that the expression of the Ci Lep gene was highest when the concentration of Lep and LEPR was 100 ng/ml,and this expression was significantly higher than that groups of 0 ng/ml,10 ng/ml and 1000 ng/ml.The expression of the Ci Lep gene in the intestine first increased and then decreased with increasing Lep concentration.When SIF were added at 0.1 μg/ m L and 1 μg/ m L,the m RNA expression of Lep and LEPR in intestinal cells of grass carp was significantly higher than that in other groups.(3)Effects of nutritional dipeptide levels,protein levels and protein sources on intestinal expression of Lep,LEPR in C.idella.After feeding the diets supplemented with different concentrations of nutritional dipeptide Ala-Gln for 4 weeks,the m RNA expression levels of Lep and LEPR were determined by q RT-PCR.The results showed that the expression level of Lep gene m RNA in brain of grass carp in Ala-Gn 0.5% group was higher than that in other groups.The study also showed that intestinal LEPR gene m RNA expression level was also affected by dietary Ala-Gn supplemental concentration,and the LEPR expression level was the highest in Ala-Gn 1.0% group.To determine the effects of different protein levels on the expression of Lep and LEPR,27%,32% and 37% protein levels diets were formulated for 4 weeks.The results showed that when dietary protein level was 37%,the expression level of Lep was significantly higher than that of dietary protein level 27%,and the expression level of LEPR was significantly higher than that of dietary protein level 27% and 32% groups.After feeding the diets supplemented with different protein sources(fish meal and soybean meal)for 4 weeks,the m RNA expression levels of Lep and LEPR genes of grass carp were determined by q RT-PCR.The results showed that the expression level of Lep in fish meal group was twice that in soybean meal group.Similarly,LEPR m RNA expression level in fish meal group was significantly higher than that in soybean meal group.To sum up,the experiments of cloning and identification of the grass carp LEPR c DNA sequence,from cellular level proves that the recombinant leptin and SIF can affect the Lep grass carp intestinal cells,LEPR gene expression,from the aspects of protein feed nutrition demonstrated different feed protein diet affect the secretion of grass carp Lep,thus affecting the grass carp intestinal LEPR expression level.This study laid a foundation for further studies on the nutritional regulation and physiological functions of leptin receptors,and also provided a theoretical basis for the optimization of grass carp feed formula.