Cloning And Expression Analysis Of Stearoyl-coa Desaturates(SCD)in Juvenile Gift Tilapia In Response To Different Temperature And Lipid

Stearoyl-CoA desaturase(SCD)is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acid(MUFA),and a key control point on regulation of lipid generation and oxidation in liver.As the data from Food and Agriculture Organization(FAO)show,the tilapia output has just reached 5446800 tons in about 100 farming countries during 2014,especially in China,the tilapia output rises to about 1450000 tons.Due to its poor cold tolerance and improper feeding,the aquaculture of tilapia suffered a great number of economic loss.GIFT tilapia is a main economic freshwater fish species farmed in the south of China,to study the SCD gene of GIFT tilapia is of great significant to increase the cold-resistance and improve the nutritional value of commercial fish.In the paper,for the first time in China,the present work successfully cloned SCD gene from GIFT tilapia,and analyzed the gene structure and expression in different growth stages and tissues.Additionally,we evaluated the effects of the fatty acids composition and rearing temperature on the growth,physiological parameters,the composition of fatty acids and SCD gene expression and activity of GIFT tilapia.It was composite explored from the nutrition,environment and molecular level,the results would be benefit to provide a basis of fatty acid regulation and rearing healthy and high nutritional value tilapia.1.Cloning and sequence analysis of SCD gene of GIFT tilapiaThe stearoyl-CoA desaturase cDNA in GIFT tilapia(Oreochromis niloticus)was cloned by RT-PCR and RACE.The complete cDNA sequence of GIFT tilapia SCD,which contained a 173bp 5'UTR,a 152bp 3'UTR and a 1008bp open reading frame(ORF)encoding 335 amino acids.Its has high similar with Nile tilapia and Oreochromis mossambicus in GenBank,the Gene sequence homology reach the 99%and 98%,respectively and the amino acid sequence reach the 99%and 97%,respectively.Compared with other fish,the gene sequence similarity was more than 83%and the amino acid sequences homology was more than 79%.It has high genetic similarity with other fish SCD gene which means the gene in the highly conserved among species.2.The spatial and temporal expression and tissues expression of SCD gene In the paper,the expression of SCD gene on zygote and embryonic development(after fertilization 1h,12h,24h,48h,72h,96h),fish larve stage(after hatch 1d,5d,10d,15d,20d,25d,30d)and in different tissues in male and female tilapia(blood,liver,spleen,kidney,heart,foregut,hindgut,gonad,brain,skin,muscle,gill).It had high level SCD expression after fertilization 1h and 12h,and decreased at 24h,and increased slightly at 48h,then gradually decreased to the lowest at 96h.In the early time of fish larve stage(after hatch 1-15d),the expression of SCD gene had a low level,and increased gradually.On the 20d after hatch,the expression of SCD significantly increased,and decreased firstly then increased.It means in different development stages,GIFT tilapia need different fatty acids,thus resulted in different expression of fatty acid metabolism enzyme level,which can provided a theoretical basis of fatty acid nutrition for reproduction and fry breeding.We also used quantitative real time PCR(qPCR)to examine SCD expression both in male and female.SCD mRNA was differently expressed in different tissues,it highly expressed in liver,and followed by spermary in male but muscle in female,however,it was lowest in heart in both male and female.The results showed that the differences on fatty acids metabolism between the male and female tilapia,and can benefit for further research on the regulation and synthesis way of UFA of tilapia.3.Study on the expression of temperature on SCD gene in GIFT tilapia We evaluated the effect of three different temperatures on the regulation of SCD gene expression and the composition of fatty acids in muscle,moreover we also tested the growth,blood parameters and serum biochemical parameters at 22,28 and 34 ?,repectively.At the end of the trial,the final body weight of juveniles reared at 28? was higher than that of fish reared at 22 or 34?,FCR and PER were also higher at 28?.The fatty acids composition of muscle tissue differed significantly(P<0.05)among the treatment groups.The content of SFA decreased with decreasing temperature whereas the UFA content increased.We observed high levels of PUFA,particularly n-3PUFA,in fish reared at the lower temperature.Rearing at low temperature significantly(P<0.05)increased the expression-and activity of the SCD gene.Increased SCD activity and gene expression can increase the biosynthesis of MUFA in GIFT tilapia muscle.Additionally,cold acclimation can decrease the content of TC and TG in GIFT tilapia,thereby altering their fatty acid composition to increase cold tolerance.4.Study on the expression of different lipid on SCD gene in GIFT tilapiaIt will effect on both the growth and physiology that adding different oil in the diet,mainly due to the different fatty acid composition and different fatty acid utilization of different fish.In this study,the diet were supplemented with coconut oil(enriched in SFA),olive oil(enriched in MUFA),fish oil(have a balance fatty acid composition)and fish+soybean oil(1:1)(enriched in PUFA)for GIFT tilapia rearing for 60 days.Through analyzing the growth index,the diet supplemented with fish oil which had a balanced fatty acid,get a better growth,and it had influence on physiological indexes of blood and liver by adding different lipid.It would rising the content of TC and TG by adding SFA,while the PUFA can reduce the fat deposition.Adding PUFA also can enhance immunity,improve the activity of antioxidant enzymes and reduce the oxidative damage.The composition of fatty acid in dietary can directly affect the muscle fatty acid composition.The content of SFA and UFA in the fish positively correlated with the content of SFA and UFA in the dietary,respectively.In addition,increasing the content of PUFA in the diet can decreased the expression and activity of SCD gene,while the SFA showed an opposite character.Therefore,balancing the composition of fatty acids and increasing the PUFA appropriately can help to achieve a good growth performance and decrease the blood lipid deposits.