| With the improvement of living standards,people’s requirements for the quality of duck meat have also been continuously improved.The type and content of fatty acids in feed directly affect the fatty acid composition in duck meat,thereby affecting the quality of duck meat,especially the balance of n-6/n-3 PUFA(polyunsaturated fatty acids)is of great significance to human health.However,the main gene affecting the composition of n-6 PUFAs and n-3 PUFAs in duck meat has not been identified.In this study,the changes of n-6 PUFA and n-3 PUFA in duck pectoral and leg muscles were determined at 56 days of age after duck fed duck fat and flax oil,respectively.At the same time,using duck fat group as the control group and flax oil group as the experimental group,the differences in gene expression in duck pectoral muscle tissues were analyzed by transcriptome and proteome sequencing,and the key genes affecting the ratio of n-6/n-3 PUFA in duck pectoral muscle were screened.In addition,the induction differentiation model of ALA(linolenic acid)replacement of OA(oleic acid)was established,and the effects of ALA replacement of OA on intramuscular fat cells(IMF)cell activity,cell proliferation,lipid deposition and key genes of fatty acid metabolism were investigated,and PLIN2 was interfered with under this model,so as to explore the effect of PLIN2 on the ratio of n-6/n-3 PUFA,which provided a theoretical basis for studying the synthesis mechanism of n-6 PUFA and n-3 PUFA.The main findings are as follows:Experiment 1:Effect of dietary supplementation of linseed oil on plasma biochemical parameters and fatty acids in duck muscleIn this experiment,in order to explore the effect of adding flax oil to the diet on duck muscle fatty acids,the first group fed different diets for ducks,of which the control group was fed a basic diet+2%duck fat(duck fat group),the second group was fed a basic diet+2%duck fat for the first 14 days,a basic diet+2%flax oil was added for 14 days(mixed feeding group),and the third group was fed a basic diet+2%flax oil(flax oil group).The fatty acid composition of duck fat,flax oil and duck fat and flax oil after adding duck oil and flax oil was detected by gas chromatography-mass spectrometry,and it was found that the content of ALA(α-linolenic acid)in the feed with 2%flax oil was about 9 times that of the diet with 2%duck fat,and the n-6/n-3 PUFA ratio was greatly reduced.The plasma biochemical levels were measured by the matching kit,and the results showed that the mixed feeding group and the combined linseed oil group significantly reduced the triglyceride(TG)content(P<0.05)and significantly increased the total cholesterol(TC),high-density lipoprotein(HDL-C)and very low density lipoprotein(VLDL-C)(P<0.01).The determination of fatty acids of duck leg muscle and pectoralis muscle,it was found that n-3 PUFA in the mixed feeding group and the linseed oil group increased the content of n-3 PUFA in the leg muscle and reduced the content of n-6 PUFA in the flax oil group(P<0.01),and n-6/n-3 PUFA in the mixed feeding group and the linseed oil group significantly reduced the n-6/n-3 PUFA in the leg muscle(P<0.01).The content of n-3 PUFA in the pectorals was significantly increased in the linoleum group(P<0.01).Treatment in mixed feeding and linoleum significantly reduced n-6/n-3 PUFA in pectoral muscle(P<0.01).The contents of ALA,EPA and DHA in the linseed oil group were significantly increased(P<0.05).Therefore,the addition of flax oil to the diet can affect the plasma biochemical indicators and fatty acid composition of duck muscle.Experiment 2:Screening of key genes composed of n-6/n-3 PUFAIn this experiment,transcriptome and proteome sequencing were performed on the pectoral muscle tissues of duck fat group and linole oil group,and bioinformatics was used to analyze differential genes and proteins,and a total of 367 differential genes(197 significantly upregulated and 170 significantly down-regulated)and 1740 differential proteins(606 significantly up-regulated,1134 significantly down-regulated)were screened.GO and KEGG enrichment analysis was performed on the differential genes and proteins,and transcriptome GO analysis showed that these differential genes were mainly enriched in the metabolic process of phosphate-containing compounds,phosphorus metabolism process,signaling receptor binding process,etc.KEGG analysis found that these differential genes were mainly enriched in FoxO signaling pathway,MAPK signaling pathway,adipocytokine signaling pathway,PPAR signaling pathway,linoleic acid metabolism,fatty acid degradation,fatty acid metabolism and other fatty acid-related pathways.Proteome GO analysis found that differential proteins were mainly enriched in fatty acid biosynthesis process,lipid metabolism process,fatty acid β-oxidation,long-chain fatty acid transport,fatty acid metabolism process,etc.,and KEGG analysis found that these differential proteins were mainly enriched in fatty acid biosynthesis,fat digestion and absorption,adipocy cytokine signaling pathway,fatty acid metabolism,fatty acid degradation,AMPK signaling pathway and other signaling pathways related to lipid metabolism.Then,the differential proteins were screened for association interactions with PLIN2,CD36,FASN,ATP11C,and ACSLs.Finally,venn analysis of differential genes and differential proteins enriched into the GO and KEGG pathways related to fatty acids found that there were 2 genes(CD36,PLIN2)co-expressed in transcriptome KEGG and proteome GO and KEGG,It is speculated that PLIN2 and CD36 may be key genes affecting the composition of n-6/n-3 PUFA.Experiment 3:Effect of key genes on fatty acid metabolism under in vitro induction model20%,40%and 60%of OA(oleic acid)was replaced by ALA as a replacement agent for oleic acid in the fat-induced differentiation model,and the optimal replacement concentration was detected by CCK8,EDU,qRT-PCR,and oil red O detection technology,and the in vitro induced differentiation model of duck intramuscular adipocytes(IMF)was constructed.The results showed that the cell activity increased and then decreased after ALA replacement OA,and the cell activity of 40%ALA replacement group was the highest at 6 days of cell induction.It was found that the 40%ALA replacement group had a higher proliferation capacity than the control group when induced for 6 days,and the lipid deposition capacity of the 40%linolenic acid replacement group was significantly lower than that of the control group(P<0.01).In addition,it was found that ALA replacement could increase the expression of PLIN2 and ELOVL2,and under the condition of 40%linolenic acid replacing OA,interference with PLIN2 expression could reduce the lipid deposition capacity of IMF and reduce the expression of ELOVL2,a key gene for DPA synthesis of DHA.In summary,adding flax oil to feed can change the composition of n-6 PUFA and n-3 PUFA in duck pectoral muscles,and reduce the ratio of n-6/n-3 PUFA.The key genes PLIN2,CD36,FASN,etc.affecting this composition were jointly identified by transcriptome and proteome.In vitro induced differentiation model experiments,it was shown that replacing OA with ALA could affect the expression of IMF lipid deposition and fat metabolism-related genes,while PLIN2 could also affect the expression of ELOVL2,a key gene for DPA synthesis of DHA.These results provide a theoretical basis for studying the synthesis mechanism of n-6 PUFA and n-3 PUFA,and provide an effective target for breeding high-quality duck breeds with balanced n-6/n-3 PUFA. |
