Integrated Analysis To Identify Candidate Functional Genes For Fat Formation And Deposition In Ducks

Lipogenesis and fat deposition are complex traits in duck.On the one hand,adipose tissue plays an important role in maintaining body homeostasis,while,on the other hand,ectopic excessive deposition of fat is closely related to feed conversion rate and organism diseases.In addition,the requirements for fat content among muscle,subcutaneous and abdominal tissues vary much.These key issues in the industry needs to be solved urgently.In this study,Cherry Valley ducks,Crested duck and their F2 resource populations with significant differences in fat deposition were selected as target subjects.And we screened candidate genes affecting formation and deposition of fat in ducks at the level of group phenotype,whole genome,transcriptome,etc.Integrating the data of genome-wide association analysis?GWAS?and RNAomics,joint meta-analysis was used to further identify key functional genes related to formation and deposition of in order to screen out a series of candidate functional genes that affect ducks' lipogenesis and fat deposition.This study provides a reference for comprehensively revealing the molecular regulation mechanism of lipid formation and deposition in different duck breeds and different tissues,and reducing the harm caused by ectopic deposition of fat.The main findings are as follows:1.Researches on establishment of duck pre-adipocyte induction system and its lipid-forming capacityPrimary pre-adipocytes were isolated from Cherry Valley ducks subcutaneous adipose tissue at embryonic day 20?E20?by collagenase digestion.Adipocytes cannot synthesize fatty acids on their own in poultry,so that it is necessary to add exogenous fatty acids to stimulate fat formation.In this study,oleic acid was used as a fat-inducing fatty acid in duck primary pre-adipocytes.We investigated the role of oleic acid in the differentiation of pre-adipocytes by adding different formulations of oleic acid?100 ?M,300 ?M,600 ?M?into the basic formula consisting of 10?g/?L insulin,1 ?m/L DEX,0.5 mmol/L IBMX,1 ?M rosiglitazone.Through phenotypic qualitative observation and quantitative determination,it was found that after 2 days of induction,some small lipid droplets resembling a bright spot was in the visual field and stained red by oil red O staining,and after 4 to 6 days of induction,small lipid droplets gradually accumulated,the volumes of which become larger from small ones,and spindle-shaped pre-adipocytes begin to recede.Eight days after induction,the pre-adipocytes differentiate into round adipocytes.The cytoplasm is filled with a large number of lipid droplets.The nucleus is not located the center but on one side,and the fat cells develop into oval or round mature adipocytes.At this time,almost all cells are stained red,and the content of triglyceride,the main component of lipid droplets,gradually increases with the increasing induction time.At the same time,it was found that when the amount of oleic acid added was 300 ?M,the fat content of pre-adipocytes was the highest,and within a certain range,the adipogenic amount gradually increased as the concentration of oleic acid increased.When it was increased to 600 ?M,oleic acid inhibits fat formation.Based on induction efficiency and phenotypic detection,the optimal induction system was finally established:10 ?g/uL insulin,1 ?m/L DEX,0.5 mmol/L IBMX,1?M rosiglitazone,and 300 ?M oleic acid,which successfully induced duck pre-adipocytes as adipocytes.A significant difference in intramuscular fat,abdominal fat,or subcutaneous fat was all found between big-sized meat duck-Cherry Valley Duck and small-sized meat duck-Crested Duck.in production.It is inferred that the adipogenic capacity of pre-adipocytes may be significant different.We isolated Cherry Valley Duck and Crested Duck embryos for 20 days?E20?subcutaneous fat pre-adipocytes.After 3 days' induction in vitro,it wasfound by the detection of CCK8 that Cherry Duck Valley either in the proliferation of pre-adipocytes or the TG content was higher than that of Crested Duck,indicating the strong lipogenesis capacity of Cherry Duck Valley.2.Whole transcriptome sequencing screening key functional adipogenic gene of duck adipocyteIn order to reveal the key candidate genes for duck adipogenesis,duck primary subcutaneous preadipocytes were isolated from subcutaneous adipose tissues of Cherry Valley ducks at embryonic day 20?E20?.On the basis of the isolation and induction model of duck pre-adipocytes in vitro described above,high-throughput sequencing was performed on the third day of induction to screened differentially expressed mRNA,miRNA,lncRNA,and circRNA during adipogenesis.There were 4702 differentially expressed mRNAs?2606 genes were significantly up-regulated and 2096 genes were significantly down-regulated?and 839 differentially expressed IncRNAs?58 lncRNAs were significantly up-regulated and 781 lncRNAs were significantly down-regulated?,141 differentially expressed circRNAs?66 circRNAs significantly upregulated and 75 circRNAs were significantly down-regulated?,59 differentially expressed miRNAs?32 miRNAs were significantly up-regulated,27 miRNAs were significantly down-regulated?after 3 days induction of duck preadipocytes.Through functional annotation of GO and KEGG,it was found that the differentially expressed genes were mainly enriched in the process of biosynthesis of lipid-associated unsaturated fatty acids and fatty acids,and cell metabolism.Pathways analysis showed that fatty acid metabolic pathways,and steroids biosynthesis,PPAR? signaling pathways,etc were significantly enriched.Nine mRNAs,seven lncRNAs,seven circRNAs,and fifteen miRNAs were selected to verify the accuracy of the sequencing results on the samples of Cherry Valley ducks by RT-qPCR.All the expression trends of the RNAs were consistent with the transcriptome sequencing results.The results confirmed the reliability of the transcriptome sequencing results.Through bioinformatics prediction of targeted miRNAs in differentially expressed IncRNAs and circRNAs,a competitive endogenous?ce?regulatory mechanism of lncRNAs/circRNA-miRNA-mRNAs was constructed.Among them,these playing key roles include XLOC₀14103-miR-181b-5p-MMP13,XLOC₀01895-miR-122-5P-KLF2,circ₀003172-miR-15c-5p-LYZ,circ₀009143-miR-206-FAS and circ₀003172-miR-219b-KLF2,etc.,This study laid a foundation for further study of lipid mechanism in duck adipocytes and screened the crucial RNA related to adipogenesis.We have performed functional verification of genes that have direct effects on adipogenesis,including DLK1,PPARy,FAS,C/EBPa,FABP4,and LPL.We found that DLK1 acts as a pre-adipocyte factor decreased with adipogenesis in preadipocytes.The highest expression levels of FABP4 and LPL genes occurred after 10 days of induction,while the expression of C./EBPa and FAS peaked at 8 days after induction.With the prolongation of differentiation time,the expression of PPARy gradually increases and peaked on the 6th day after induction.The above indicates that DLK1,PPARy,FAS,C/EBPa,FABP4,and LPL all play an important role in the formation of adipocytes.In order to further study PPARy,a star gene that affects poultry fat traits,and at the same time to certificate the sequencing data above and cell-level validation results,and to figure out the key role that PPARy plays in duck fat formation,therefore,we,knocked down siRNAs that specifically interfered with PPARy.After 48 h of interference with PPARy,it was found that the TG content of primary adipocytes decreased,and the mRNA expression level of the related adipogenic genes also showed a decrease.Among them,the expression levels of the transcription factors C/EBPa and FABP4 decreased but the difference was not significant,and LPL The expression level was significantly decreased?P<0.05?.It was shown that PPARy can indeed affect duck fat formation and play a key role.It is one of the important candidate genes.In the previous experiments,it was verified that there was a significant difference in the adipogenic capacity between Cherry Valley Duck and Crested Duck.To explore the molecular mechanisms of fat differentiation in different breeds,we compared and analyzed the differences in miRNAs and key mRNAs between Cherry Valley Duck and Crested Duck.First,the key miRNAs affecting crested ducks' adipogenesis were screened from adipocytes and preadipocytes in crested ducks after induction for 72 hours.As a result,there were 105 differentially expressed miRNAs?50 miRNA were significantly up-regulated and 55 miRNA were significantly down-regulated?in Crested Duck,and 7 miRNAs were selected to verify and the results were consistent with the sequencing results.Among the 105 differential expression miRNAs,a total of 37 miRNAs were shared by Cherry Valley ducks and Crested Ducks.In the 37 miRNAs that overlapped,31 showed consistent trends in the two breeds.We speculate that these miRNAs may play an indispensable role in the process of fat production in ducks.22 miRNAs were specifically expressed in Cherry Valley ducks,and 68 were specifically expressed in Crested ducks.After target gene prediction,There were 419 genes specifically expressed in the cherry valley adipose process in combination with the differential genes screened by mRNA-seq in Cherry Valley Duck.They are significantly enriched to signal pathways such as fatty acid elongation,biosynthesis,and metabolism.Furthermore,the key genes for adipogenesis,such as PPARy,FAS,C/EBP?,FABP4,and LPL,were validated on two varieties.It was found that the expression of the four genes in the adipocytes of Cherry Valley ducks except LPL was significantly higher than the Crested Duck.Once again,it was verified that the Cherry Valley ducks had higher fat deposition ability than the Crested duck at the molecular level.3.Genome-wide association analysis identifying candidate genes related to intramuscular fat and abdominal fat depositionIn order to further identify the key genes of duck fat formation,on the basis of cell-level studies,we analyzed the key genes or markers affecting fat deposition in ducks through genome-wide association analysis at the individual level for production applications.Firstly,the F2 resource group of Cherry Valley duck???×Crested duck???was established.After breeding for 42 days,304 animals were slaughtered and collected.Blood was collected and the weight of abdominal fat and intramuscular fat content of pectoral muscle were measured.The genome DNA was re-sequenced by 10X whole genome and genome-wide association analysis was performed combining phenotypic values.The results showed that the intramuscular fat ranged from 1.26%to 1.503%with a mean of 1.387%and the weight of abdominal fat ranged from 4 to 51 g with an average of 27.622 g.The phenotypic value of the traits was in a normal distribution.The Bonferroni multiple test calibration method was used to determine the P value.When the significance threshold was 5?-log10P?,14 extremely significant SNPs were co-localized for the intramuscular fat content of pectoral muscles.According to the database information in NCBI,Ensembl,KEGG,Uniprot,and GeneCards,the function of genes in a single LD?20 kb?around the SNP of the significant locus were annotated used bioinformatics and comparative genomics.25 genes including BARHL2,SLC22A16,THBD,OGDH.CD93,Ctgf.etc were found and mainly involved in activity of DNA-binding transcription factor,neuronal migration,kinase activity adjusting the body's metabolism of nutrients and balance.The weight of abdominal fat traits were mapped to 12 extremely significant SNPs.After annotation,a total of 48 genes including MICALL1,GALR3,SOX10,EIF3L,and POLR2F.After functional annotation of all selected adipose-related genes,it was found that PGDFB and ATF4 were significantly enriched to signal pathways such as MAPK and hormone metabolism.MAPK signaling pathway also plays a role in regulation of intramuscular fat and abdominal fat mass.At the same time,the PI3K-AKT pathway involved by LAMA4 regulates the production and metabolism of intramuscular fat through glycolysis and gluconeogenesis,and is mediated by PDGFB.Inhibition of the PPAR? pathway regulates fat deposition by interacting with ATF4 via the SOS signaling pathway and Erkl/2.4.Joint analysis of GWAS and transcriptome to identify lipogenic genesThe results of integrated transcriptome sequencing and whole-genome association analysis revealed that the 73 genes in the 26 SNPs of the genome-wide association analysis and the 4702 differentially expressed genes screened in the transcriptome were jointly analyzed and found that there were 18 intersectional genes?LAMA4,SYNJI,CD164,CDK19,SLC22A16,RPF2,GTF3C6,AMD1,SSTR4,CARD11,SH3BP1,GCAT,MICALL1,POLR2F,JOSD1,RPL3,PDGFB,MGAT3?totally.Through functional annotation and pathway analysis,the key genes PDGFB,MGAT3,LAMA4,SLC22A16,AMD1,and CD 164,which affect duck fat formation and deposition,were screened.Through the pathway analysis of the selected genes,it was found that PDGFB is closely related to the mTOR signaling pathway.Therefore,the following hypothesis is proposed:PDGFB may inhibit the mTOR signaling pathway through the initiation of PI3K/Akt signaling pathway,further interact with PPARy signaling pathway,and regulate the production of fat together with mRNA,miRNA,lncRNA and circRNA.In summary,the process of fat deposition is complex.MGAT3,LAMA4,SLC22A16,PDGFB,AMD1,and CD164 have large effects on adipogenesis at both SNP and RNA levels.The BARHL2 and ATF4 identified by genome-wide association analysis and the MMP13 and KLF2 genes screened from transcriptome analysis may be potential candidates for the formation and deposition of fat.