| Scylla paramamosian,one of the most popular mud crab species,has been widely cultured in the coastal areas of southern China.With the development of aquaculture and the tensions of feed raw,contradictions of aquatic animal feed supply and demand are sharpending.In order to alleviate the international shortage of fish oil resources,this study was conducted to investigate the effects of dietary fish oil replacement by soybean oil on growth performance,proximate composition,fatty acid composition and biochemical parameters,fatty acid synthesis-related genes,fatty acid transport-related genes and catabolism-related genes expression in mud crab.And by the key genes Δ6 Fad promoter of high unsaturated fatty acid synthesis as the breakthrough point,Screening and cloning main transcription factors.On basis of these studies,it was aimed to investigate mechanisms involved in regulation of dietary fish oil replacement by soybean oil on lipid metabolism of mud crab,Scylla paramamosain.Study on lipid nutrition metabolism of mud crabwill be helpful for providing reference data of research and development of artificial feed on mud crabs,and providing theoretical data for crustacean lipid metabolism.Studies on the nutritional requirement will be very important to develop nutritionally balanced and cost-effective diets for mud crabs.The results and conclusions are summarized as follows:1.The effect of dietary fish oil replacement by soybean oil on the growth performance and biochemical composition in mud crabs.With the fish meal,casein,complex vitamins and complex minerals as basic feed materials,six isonitrogenous(45 % crude protein)and isolipidic(9.5 % crude lipid)experimental diets were formulated to contain different soybean oil(SO)levels with fish oil(FO)replacement by 0 %(SO0),20 %(SO20),40 %(SO40),60 %(SO60),80 %(SO80)and 100 %(SO100),respectively.There were six treatments,each treatment with 60crablets(initial average weight 20.19 ± 0.82 g),and crablets were apparent satiation for 10 weeks.The results showed that weight gain rate and specific growth rate of mud crab decreased with the increase of soybean oil substitution level in the feed,the SO60,SO80 and SO100 groups were significantly lower than other groups(P < 0.05).overall consideration that the optimal replacement level in mud crab diet should not exceed 40%;The crude lipid content of SO60,SO80 and SO100 groups in hepatopanpancreas increased significantly(P<0.05),crude protein content of SO0 group was significantly higher than that of SO20,SO40 and SO60 groups.The crude lipid content in muscle tissue generally decreased with the increase of SO substitution level.EPA in hepatopancreas gradually decreased with the increase of SO substitution.The lowest value was found in the SO100 group,and there was no significant difference in DHA content among treatment groups(P>0.05).In muscle,the EPA content in SO0 and SO20 groups was significantly higher than that other groups(P<0.05).DHA content in the SO0 group was significantly higher than SO80 and SO100 groups(P<0.05).The content of HUFA in muscle and hepatopancreas was negative correlated with the content of C18:2n-6 in feed fatty acids,while the content of C18:2n-6 and HUFA in hepatopancreas was positive correlated with that in muscle.The hepatic LPL and HL activities and TG concentration in mud crab did not show any statistical difference among groups(P > 0.05).2.The effect of dietary fish oil replacement by soybean oil on the lipid metabolism genes in mud crabs.With increasing dietary SO level,FAS and ACC genes expression in the hepatopancreas significantly increased,FAS gene expression were up-regulated about2.48-fold and 2.23-fold,and ACC gene expression up-regulated about 2.25-fold and2.53-fold compared with that in the SO0 group respectively when the replacement ratio of FO by SO up to 80 % and 100 %(P < 0.05).In muscle,no significant differences in the expression of FAS gene were found among all treatments(P > 0.05),ACC gene expression in SO80 and SO100 group were up-regulated about 1.98-fold and 1.71-fold compared with that in the SO0 group respectively(P < 0.05);the hepatic and muscles transcript level of FABP3 in SO0 and SO20 groups were significantly lower than that other groups(P < 0.05).The FATP gene expression increased first and then decreased with the increase of soybean oil replacement ratio,and the SO60 and SO80 groups were significantly higher than other treatment groups(P < 0.05),which significantly lower in SO0 group than other treatment groups(P < 0.05)in muscle,and FABP9 expression increased overall with the increase of SO substitution;In hepatopanpancreas,LPL gene expression level was significantly reduced when the replacement ratio reached 80% and 100%(P < 0.05),and CPT1 gene expression level was significantly reduced when the replacement ratio reached 60%,80%and 100%(P < 0.05).In muscle tissue,the m RNA expression level of CPT1 gene in the SO0 group was significantly lower than that in the SO40,SO60,SO80 and SO100 groups(P < 0.05).3.cloning and bioinformatics analysis of Δ 6 Fad gene promoter in mud crabs.The promoter of Δ6 Fad was cloned with 2044 bp in length,including the second exon70bp(partial sequence),the first intron area 228 bp,the first exon area 126 bp,the first exon upstream sequence 2833 bp.Analysis on the promoter prediction website showed that the sequence had promoter characteristics and elements of transcription factors might be related to lipid metabolism,including: NF-1、HNF-4、CEBPB、SREBP、Sp1、AP1 and P53,a 102 bp Cp G island was found at-958 ～-856 bp site.4.cloning and bioinformatics analysis of SREBP-1 in mud crabs.The full-length c DNA of SREBP-1 was 3361 bp(excluding poly(A)tail),which included a 5’-terminal untranslated region(UTR)of 123 bp,a 3’-terminal UTR of 118 bp and an open reading frame(ORF)of 3210 bp encoding a polypeptide of 1039 amino acids.The deduced protein sequence of SREBP-1 displayed a b HLH-zip domain,that is the typical structure of SREBP.Alignment analysis of the deduced mud crab proteins with their orthologs in other species showed that mud crab SREBP-1 shared high sequence identity with Procambarus clarkii(61.17 %),shared 32.57 % and 31.84 % identity with those of Salmo salar and Danio rerio respectively,and 11.07-33.30 % identity with Srebp-1 from human,mouse and chicken.Mud crab SREBP-1 m RNA was detected in all studied tissues,but the expression levels varied among the tissues.Relatively high expression level of SREBP-1 was observed in eyestalk and cranial ganglia(P < 0.05),and moderate levels inthe hepatopancreas,heart,gill and intestine.The lowest abundance in muscle,stomach and thoracic ganglia.5.The effect of soybean oil replacing fish oil on the Δ6 Fad and SREBP-1 genes in mud crabs.The Δ6 Fad genes expression showed increased tendency with the increased dietary SO levels,which up-regulated about 2.76-fold and 2.58-fold compared with SO0 group respectively(P < 0.05),when the replacement ratio up to 80 % and 100 %.In muscle,Δ6Fad gene expression is the highest,And a significant difference was found compared with SO0 group(P<0.05);Compared with SO0 group,SREBP-1 transcripts in hepatopanpancreas increased about 2.25 and 2.87-fold respectively when 80 % and 100 %of dietary FO was replaced with SO(P < 0.05).SREBP-1 transcriptional levels in muscles showed increasing tendency with the increased replacement of dietary FO by SO,which up-regulated about 2.03-fold,1.86-fold and 2.13-fold respectively when the replacement ratio up to 60%,80 % and 100 %(P < 0.05).Different proportion of soybean oil to replace fish oil feed on mud crab in the hepatopancreas Δ6 Fad,the influence of the amount of SREBP-1 gene expression than in muscle tissue.Summary,the above results showed that:(1)The replacement of fish oil with different proportions of soybean oil in the feed has a certain impact on growth,and has a significant impact on the composition of hepatopancreatic body and fatty acids;On the basis of 8% oil,the proportion of soybean oil replacing fish oil should not exceed 40%.(2)The overall expression level of fatty acid synthesis and transport related genes increased,and the expression level of lipodieresis related genes was down-regulated.The expression level of each gene in muscle tissue was consistent with that in hepatopanpancreas as a whole.(3)Δ6 Fad promoter region contains a conservative NF-Y,SREBP binding sites,on the basis of the cloned transcription factor SREBP-1,and study the effects of soybean oil replacing fish oil.Δ6 Fad and SREBP-1 gene expression is higher with the percentage of soybean oil to replace fish oil rises,and the influence is larger in the hepatopancreas compared with that in muscle tissue. |
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