| Redlip mullet is an important economic species in low-salt water and mariculture in China and now it is one of the new species of sea water and fresh water in Jiangsu coastal area.Little information is available in the production of formula feed and the diets are usually produced based on the requirements for other omnivory fish.Sometimes,mullets may be harvested with extensive fat deposition in fish body(especially in abdomen).Fat,protein and carbohydrate are important nutritional ingredients of mixed feed.Their imbalance can lead to lipid metabolism disorder and excessive accumulation of fat.Its occurrence and development are closely related to lipid metabolism key factors.The paper,by means of molecular biology and bioinformatics,cloned for the first time the lipid metabolism key genes of PPARa,PPAR?,LPL and FAS.It also investigated the gene expression pattern in different tissues in mullet.On this basis,this paper studied the effects of dietary lipid and protein levels on fat deposition and expression of lipid metabolism related genes in mullets.The results may provide necessary reference for the development of compound feed and the regulation of lipid metabolism in mullet.1 Effects of dietary lipid levels on the growth,blood and liver biochemical indexes of mulletWith singular factor density gradient method,six iso-nitrogenous(30.7±0.1%crude protein)and iso-energetic(22.3±0.1 MJ/kg gross energy)diets were formulated.Increasing amounts of fish oil were incorporated to provide graded lipid levels(2.04%?4.83%?7.47%?9.79%?12.01%?14.59%on a dry matter basis).Juvenile mullets(initial weight 9.5±0.3 g)were distributed randomly into 18 barrel-shape tanks,with 30 fish stocked in each tank.Each experimental diet had three replicates.At the end of the 60-day feeding trial,fish were starved for 24 h.Then the samples were collected for analysis.The results were shown as follow:There was no significant difference in fish weight gain rate(WGR)and feed conversion ratio(FCR)between dietary lipid level of 7.47%and 9.79%(P>0.05).However,the above two groups had significantly higher WGR and lower FCR than other groups.About the protein efficiency ratio(PER),no significant difference was found between group 9.79%and 7.47%(P>0.05),but the PER of the former group is significantly higher than other groups(P<0.05).The polynomial regression model was used to estimate the optimal dietary lipid level based on WGR.The relationship between WGR and dietary lipid level was best expressed by the second-order polynomial regression analysis(y=-0.8238x2+14.95x+162.51;R2=0.7751)(quadratic,P<0.001).The optimal dietary lipid level was determined to be 9.1%for maximum WGR.Along with the increase of dietary lipid level,activities of glutamate pyruvate transaminase(GPT)and glutamic oxalacetic transaminase(GOT)of plasma raised significantly(linear,P<0.05).Dietary lipid level had no significant effect on the content of triglyceride(TG),cholesterol(CHO),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C).With the increase of lipid level,plasma catalase(CAT)increased first and then decreased(quadratic,P<0.001),and total superoxide dismutase(T-SOD)showed the same trend(quadratic,P<0.001).Plasma total antioxidant capacity(T-AOC)decreased with the increasing of dietary lipid level(linear,P=0.014).On the contrary,the content of malonaldehyde(MDA)significantly increased with the increase of lipid level(linear,P<0.001).In view of that,fish fed 7.47%and 9.79%lipid had good production performance and liver biochemical indices,so suitable lipid level ranged from 7.5%-10%for juvenile mullet should be recommended.2 Effects of dietary lipid levels on mullet body index,fat deposition and fatty acid compositionFeeding Regimen was the same as that of the One.Vertical somatic index and hepatopancreas somatic index increased first and then decreased with the increasing of dietary lipid level(quadratic,P<0.05).There were no significant differences among the four groups in the lipid level of 4.83%,7.47%,9.79%and 12.01%(P>0.05).Lipid content in muscle and whole body positively correlated with dietary lipid level(linear,P<0.05).Proportion of fat in muscle(20.30%?24.31%),liver(2.43%?2.95%)andmesenteric fat(11.33%?12.47%)out of the total body fat did not change significantly among treatments(P>0.05).No significant difference in fat accumulation level was found among groups in the above three tissues(P<0.05).Polyunsaturated fatty acids were enhanced with increasing dietary lipid levels in all three tissues examined,as well as whole body analysis.The ratio of n-3/n-6 showed a significant increase with increasing fish oil levels in three tissues and whole body analysis(P<0.05).As compared with EPA content in three tissues and whole body lipids,higher DHA content was found in each experimental treatment.3 Molecular cloning and tissue expression analysis of several lipid metabolism related genes of redlip mulletThe PPARa,PPARy,LPL and FAS cDNA sequences were cloned from mullet by using reverse transcriptase-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)method.In addition,the tissue expression of above four genes were determined by real time-quantitative PCR(RT-qPCR)technology.(1)Molecular cloning and tissue expression analysis of PPARaThe obtained complete PPARa cDNA was 2409 bp in length encoding 478 amino acids(GenBank accession number:KJ848472.1).The PPARa protein was predicted to contain 4 domains,including the hypervariable region in N-terminus,DNA-binding domain(DBD),flexible hinge domain,and ligand-binding domain(LBD)in C-terminus.Complete AA sequence alignment showed that the identities of amino-acid between mullet PPARa and PPARa gene of other species were very high.Some important functional sites of PPARa were highly conserved throughout the evolutionary process.PPARa was expressed in all the tested tissues,and the expression of PPARa mRNA occurred predominantly in liver,followed by brain,stomach,skin,spleen,mesenteric fat,gill,kidney,intestine and heart,but was weak in muscle.(2)Molecular cloning and tissue expression analysis of PPARyThe full-length cDNA of PPARy(GenBank accession number:KJ848473.1)sequence in mullet was 2985 bp,consisted of a 200 bp 5'UTR,1183 bp 3'UTR and 1599 bp ORF encoding a polypeptide with 533 amino acids.Similar to other PPARa,the PPAR? protein was predicted to contain 4 domains,including the hypervariable region in N-terminus,DBD,flexible hinge domain,and LBD in C-terminus.Sequence analysis showed that mullet PPARy shared highly identities with other vertebrates PPAR?,and the gene was also conserved in mullet.PPARy mRNA was detected in all 11 tested tissues and was expressed in mesenteric fat with the highest level,followed by intestine,gill,kidney,liver,skin,brain,spleen,stomach,while a little expression of PPARy mRNA was observed in heart and muscle.(3)Molecular cloning and tissue expression analysis of LPLThe full-length cDNA of LPL(GenBank accession number:KJ825894.1)sequence in mullet was 2395 bp,consisted of a 168 bp 5'UTR,676 bp 3'UTR and 1551 bp ORF encoding a polypeptide with 516 amino acids.LPL contained two structural regions:a N-terminal region(24-362 residues)and a C-terminal region(363-516 residues).Some important functional sites,such as the lipid binding domain and the catalytic active center(Ser-Asp-His),are highly conserved in evolution.LPL mRNA was expressed in mesenteric fat with the highest level,followed by hepatic,stomach,kidney,gill,intestine,brain,heart,spleen,while the lowest expression of LPL mRNA was observed in muscle.(4)Molecular cloning and tissue expression analysis of FASThe obtained partial FAS cDNA was 920 bp in length encoding 303 amino acids(GenBank accession number:KJ848474.1).Sequence analysis showed that mullet FAS shared highly identities with other vertebrates FAS,ranging from 74%?95%.Mullet FAS mRNA expression abundance was significantly different in all tested tissues(P<0.05).FAS mRNA expression level in brain was the highest,followed by liver and mesenteric fat,the lowest expression in muscle,and the expression of the other seven tissues was significantly lower than that of brain and liver(P<0.05).4 Effects of dietary lipid levels on expression and activities of several lipid metabolism related genes of mulletFeeding regimen was the same as that of the one.PPARa mRNA expression in liver increased significantly with the increasing dietary lipid levels(P<0.05).However,there was no significant difference in the expression level of PPARa mRNA in muscle and mesenteric fat among all groups respectively(P>0.05).PPARy mRNA expression in the liver,mesenteric fat and muscle showed an increasing trend with the promoting lipid level.However,no significant difference was observed among fish fed different diets in the three tissues.The expression level of LPL mRNA and FAS mRNA in abdominal fat and muscle tissues showed little change with the increase of dietary lipid level,and no significant differences were observed among the groups(P>0.05).The abundance of LPL mRNA in the liver increased with the increasing dietary lipid(P<0.05).The liver LPL mRNA expression was significantly higher in the group of 12.01%and 14.59%than that in the other three groups of 2.04%?4.83%and 7.47%(P<0.05).The activity of LPL in liver showed the same trend with the increase of lipid level,and the enzyme activity of the group 14.59%was significantly higher than that of group 2.04%and 4.83%(P<0.05).FAS mRNA levels in liver significantly decreased with increasing dietary lipid(P<0.05).Fish fed diets with 12.01%and 14.59%lipids had significantly lower liver FAS mRNA abundance than that with 2.04%lipid,and there were no significant differences among other three groups.Liver FAS activities showed a declining trend with increasing dietary lipid levels,and fish fed diets with 14.59%lipids had significantly lower liver FAS activities than that with 2.04%and 4.85%lipids(P<0.05).5 Fat deposition and liver antioxidation capability and expression and activities of several lipid metabolism related genes of redlip mullet subject to different dietary protein levels.Four isoenergetic experimental diets(12.68 MJ/kg)were formulated to contain protein levels of 26%?28%?30%and 32%.In the formula,the energy loss caused by the decrease of the concentration of protein was supplemented with corn starch.Triplicate groups of mullets(initial weight,92.20±4.23 g,gross weight 2300 kg)reared in freshwater pond were fed with four experimental diets.At the end of the 60-day feeding trial,fish were starved for 24 h.Then the samples were collected for analysis.The results were shown as follow:Proportion of fat in muscle(23.29%?24.98%),liver(3.30%?4.27%)and mesenteric fat(7.11%?12.75%)out of the total body fat did not change significantly among treatments(P>0.05).In the three tissues,proportion of fat in any one of the three tissues out of the total fat in the 26%protein group was also higher than that in other three groups.The activity of GSH-PX(quadratic,P=0.012)and T-SOD(quadratic,P=0.014)of liver increased first and then decreased with the increase of dietary protein level.Fish in group of 26%protein had higher MDA content in liver than that in other three groups(P<0.05).Dietary protein level had no significant influence on the activities of T-AOC and CAT(P>0.05).With the increasing dietary protein level,the expression level of PPARa mRNA in muscle decreased first and then increased,and the lowest value was obtained at dietary protein level of 30%(P<0.05).PPARa mRNA in mesenteric fat showed similar change trend to that in muscle.There were no significant differences in liver PPARa mRNA among groups(P>0.05).The expression level of PPARy mRNA in three tissues(muscle,mesenteric fat,liver)all obtained the highest value at dietary protein level of 26%,which was significantly higher than that in other groups,except for the group of protein level of 32%in muscle.Both FAS mRNA expressions and activities in three tissues decreased first and then increased with increasing dietary protein level.The expression and activity were the highest in the group of protein level of 26%,and no significant differences were observed between the group of 28%protein and that of 30%protein(P>0.05),and then a little up-regulation of expressions and activities of FAS was observed in the group of 32%protein.No significant differences in LPL mRNA expression of muscle,mesenteric fat and liver were observed among treatments(P>0.05).There were no significant differences in the activities of LPL of mesenteric fat(P>0.05),but the activities of LPL in muscle and liver showed increased trend along with the raising dietary protein levels.As stated above,the conclusions are as follow:(1)The appropriate level of lipid in diet could promote the growth and production performance,but excessive amount of it(12.01%and 14.59)might be negative for fish production performance and liver antioxidant function.Too much dietary lipid might result in excessive fat deposition in tissues(muscle and liver)and whole body,so excessive dietary lipid in diets should be avoided.With the increase of fish oil levels,PUFA in tissues(muscle,liver and abdominal mesenteric fat)and whole body,especially n-3PUFA,increased significantly.Compared with EPA concentration in three tissues and whole body lipid,higher DHA concentration was found in each experimental treatment.The proportion of fat accumulation of juvenile mullets in the three fat accumulation sites ranking from high to low were muscle,abdominal mesenteric adipose tissue and liver.(2)The coding sequences of the four genes(PPAR?,PPAR?,LPL,FAS)were highly conserved respectively.The expression level of each gene in different tissues was significantly different.PPARa mRNA was highly expressed in liver,while PPARy and LPL mRNA were the highest in abdominal mesenteric adipose tissue,FAS mRNA was highly expressed in brain,liver and abdominal mesenteric adipose tissues.(3)With increasing fat levels,the expression level of lipogenesis genes(PPARa mRNA and LPL mRNA)in the liver increased significantly,and the activity of LPL was also increased significantly.While the mRNA expression and activity of lipogenic gene(FAS)was significantly decreased.(4)With iso-energetic and iso-fat premise,the low protein group(26%)and high protein group(32%),especially the former,raised mRNA expression and activity of lipogenesis genes(PPAR? and FAS)of the three tissues(muscle,liver and abdominal mesenteric fat),and also enhanced the fat deposition of these tissues.In both groups,the expression levels of PPARa mRNA in tissues were improved.LPL activity of fish in 32%protein group was significantly higher than that of fish in 26%protein group.Fat accumulation patterns of three fat accumulation sites of juvenile mullet in protein level nutrient testing were the same with those in the lipid experiment. |
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