Transcriptomic-based Screening Of Effector Factors Regulating Fat Metabolism In Embryonic Broilers Of High-and Low-fat Type

Benefiting from the tremendous progress made in poultry breeding and scientific breeding methods,the growth rate of broilers has been unprecedentedly improved.However,the increased growth rate of broilers also caused problems such as abdominal fat deposition,which not only reduced chicken quality and feed utilization,but low-quality meat products also seriously threatened the health of consumers.Therefore,how to reduce the excessive deposition of belly fat in broilers and improve meat quality is one of the important issues that broiler producers and scientific researchers need to solve urgently.It is known that the metabolism of high-fat and low-fat broilers is significantly different;Roche 308 broiler is a common high-fat broiler,and Qingjiao Mochi is often used as a low-fat broiler model to carry out fat metabolism-related research.Therefore,in this study,Roche 308 broilers(Ross308)and Qingjiao Mochi(QJ)were used as high-and low-fat broiler research models.Based on the analysis of the differences in liver fat metabolism in the embryonic stage of high-fat broilers,transcriptomics techniques Breed characteristics and developmental change patterns of liver gene expression in broiler chickens at embryonic stage,screening of differentially expressed factors and focusing on the functions of lipid metabolism-related factors and the metabolic pathways involved,revealing the potential effect factors that regulate fat deposition in different strains of broiler chickens,and using them Lentiviral overexpression verifies the core differential expression factors.The research aims to screen the key factors that cause the difference in fat metabolism between high-fat and low-fat broilers,and provide a theoretical basis for the selection and breeding of low-fat broilers and the regulation of fat deposition in poultry.1 Comparative analysis on the changes of related factors of fat metabolism in highand low-fat broilers during embryonic periodIn this part of the experiment,Roche 308 broiler(Ross 308)and Qingjiao chicken(QJ)were used as the test subjects to analyze the nutrient content of broiler breeding eggs,the biochemical indicators of embryonic lipid metabolism and the expression levels of key lipid metabolism factors,aiming to reveal high-and low-fat broilers the expression of the key factors of fat metabolism during embryonic period differed and the regularity of the changes.There are 120 breeding eggs for Ross 308 and QJ each,each of which is set in three repeats,each with 20 breeding eggs;hatched in an incubator,and collected serum and liver at E19 and H1.Infrared spectroscopy is used to analyze the nutritional content of breeding eggs,and the colorimetric method is used to analyze the content of triglycerides(TG),total cholesterol(TC),high-density lipoprotein-cholesterol(HDL-C)and low-density lipoprotein-cholesterol(LDL-C),Realtime-PCR analysis of m RNA expression levels related to fat metabolism.The results showed that the content of TG,TC,palmitoleic acid and oleic acid in the yolk of Ross 308 broiler eggs was significantly higher than that of QJ(P < 0.05),while trans-9-octadecenoic acid and docosahexaenoic acid in the yolk the cysteine ??content in He protein was significantly lower than QJ(P < 0.05).Ross 308 broiler embryo weight,absolute liver weight and serum TG and TC contents at different embryo age stages were significantly higher than those of QJ(P < 0.05);serum TG and HDL-C contents of two broilers were significantly higher at H1 At the E19(P < 0.05).The expression levels of ACC,SREBP-1c,FAS,APOA-1,APOB,CPT-1 and PPARα in the liver of Ross 308 broilers at different embryonic stages were significantly lower than those of QJ(P < 0.05);ACC and FAS in the livers of the two strains of broiler chickens the gene expression level at H1 was extremely significantly higher than E19(P < 0.01),while APOB,PPARα,and CPT-1 gene expression levels were extremely significantly lower than E19(P< 0.01).The results suggest that the high fatty acids and cholesterol in the high-fat Ross308 broiler egg yolk may be one of the reasons for its fast growth and development and more fat deposition;and the genes related to fatty acid synthesis,fatty acid decomposition and lipid transport in the embryonic liver tissue the expressions were lower than those of Qingjiao chicken,suggesting that Ross 308 broiler has a lower lipid metabolism rate than Qingjiao chicken during embryonic development.In addition,the expression of fat-related factors in the embryonic stage of the two strains of broilers showed regular changes,that is,the decomposition of fatty acids was inhibited and the synthesis of fatty acids was strengthened after the hull.2 Transcriptomics analysis of the characteristics of the expression of factors related to lipid metabolism in the embryo of high-and low-fat broilersThis experiment uses transcriptomics to analyze the changes in liver gene expression of Ross 308 broilers and QJ at E19 and H1,and screens the differentially expressed genes involved in fat metabolism,aiming to explore the main factors that cause the difference in fat metabolism in high-and low-fat broilers.The results showed that the two strains of broiler chickens identified a total of 804 differential genes at the E19 embryonic age and858 differential genes at the H1.After Cytoscape gene network interaction analysis,compared with QJ,E19 Ross 308 has screened 9 core genes,including EDN1(down-regulated)and GHRL(up-regulated)in the c AMP signaling pathway,and ANGPTL3(up-regulated)in cholesterol metabolism.),CYP7A1(down-regulated)in bile acid biosynthesis,PCK1(up-regulated)and ADH6(down-regulated)in glycolysis/gluconeogenesis,SREBF1(down-regulated),AOX1(up-regulated)and FGF19 in pathways related to fat metabolism(Up-regulated);13 differential core genes were screened at H1,including CYP1A1(down-regulated)and STS(down-regulated)enriched in steroid biosynthesis,CYP7A1(down-regulated)in bile acid biosynthesis,glycolysis/PCK1(up-regulated),ALDOC(up-regulated),ENO2(up-regulated)and ACSS2(up-regulated)in gluconeogenesis,and HSPA2(down-regulated),HSPA8(down-regulated),PI3KR1(down-regulated),PIK3CB(up-regulated),FABP1(up-regulated)in fat metabolism-related signaling pathways And FGF19(up-regulated).The results showed that,compared with the low-fat green-footed hemp chicken,the expression of key glycolysis genes(ADH6)in Ross 308 broilers was lower,while the expression of key gluconeogenesis genes(PCK1,PI3KR1)was higher,indicating that high-fat broilers Ross 308 broilers have weak glycolytic capacity and strong gluconeogenesis.At the same time,the expression of AOX1,CHRL,ANGPTL3 and FGF19 related to liver lipid metabolism in Ross 308 broilers is higher,indicating that the above core genes are responsible for the high-fat Ross 308 The lipid deposition effect factor of broilers.The combination of FGF19,CHRL and ANGPTL3 as extracellular signal molecules and higher expression differential multiples suggests that they may be the key effector genes that regulate lipid deposition in Ross 308 broilers.3 Transcriptomics analysis regulates developmental changes of factors related to lipid metabolism in embryonic stage of high-and low-fat broilersIn this experiment,transcriptomics technology was used to analyze the changes in liver gene expression of Ross 308 and QJ broilers at different embryonic ages,to screen the common and different key factors affecting the fat metabolism of the two broilers,and to reveal the changes in the different developmental stages of chicken embryos.The results showed that from E19 to H1,3429 and 2534 differentially expressed genes were identified in Ross 308 and QJ liver tissues,respectively.Using Cytoscape comprehensive analysis,it was found that from E19 to H1,Ross 308 broiler had 7 core differentially expressed genes enriched in glycolysis/gluconeogenesis pathway(down-regulated genes GAPDH,ALDOB,FBP2 and up-regulated genes TPI1,PCK1,PDHA1,PDHB)and 14 core differentially expressed genes are enriched in lipid metabolism-related pathways(down-regulated genes SCD and up-regulated genes FASN,ACACA,SRBPF1,ACOX1,ELOVL6,ACSL1,FADS1,FADS2,ACSL5,LPIN1,ELOVL5,ELOVL1 ACSBG2);QJ has 8 core differentially expressed genes enriched in glycolysis/gluconeogenesis pathway(down-regulated genes FBP2,ALDOB,GAPDH and up-regulated genes TPI1,LDHB,PDHB,PDHA1,PCK2)and 13 cores Differentially expressed genes are enriched in lipid metabolism-related pathways(down-regulated genes DGAT2 and up-regulated genes FASN,ELOVL6,ACACA,SREBP1,FADS1,FADS2,ELOVL1,ACSL5,ELOVL2,PLIN1,FABP4,FABP1).Further research found that HGF,ANGPT1 and FGF19 are the core differentially expressed genes in Ross 308 broiler liver tissue that are different from QJ,and are significantly enriched in c AMP,PI3K-Akt and c GMP-PKG pathways,respectively,suggesting that It may be a key factor affecting the difference in embryonic lipid metabolism between high-fat and low-fat broilers.4 Study on the role of hepatocyte growth factor in abnormal lipid deposition induced by free fatty acidsThe experiment in this chapter takes chicken liver cancer cell line(LMH)as the research object,transfects LMH cells with a lentiviral vector overexpressing hepatocyte growth factor(HGF),establishes a LMH cell model overexpressing HGF,and analyzes the expression of lipid metabolism-related factors Circumstances,aiming to explore the regulation and mechanism of HGF on poultry fat metabolism.The experiment was divided into control group,CMV-NC group(no load)and CMV-HGF(overexpression)group,and the final concentration of 0.75 m M free fatty acid(FFA)was used to induce LMH cells for24 h,and the cells were collected.Colorimetric method was used to determine cell triglyceride(TG)and total cholesterol(TC)content,Realtime-PCR method was used to determine HGF gene expression level,and Western Blot method was used to analyze HGF,ACCα and p-ACCα protein expression levels.The results showed that 0.75 m M FFA treatment can significantly increase the TG content and the number of lipid droplets in LMH cells(P < 0.05).Compared with the control group,FFA induction can significantly reduce the expression level of HGF protein in LMH cells(P < 0.01).Compared with the control group and the CMV-NC group,the HGF gene expression and protein expression levels of the CMV-HGF group were significantly up-regulated(P < 0.01),and the content of TG and TC in the LMH cells of the CMV-HGF group was significantly reduced(P <0.05),suggesting that HGF can alleviate excessive lipid accumulation in LMH cells induced by high fat.Compared with the control group,the expression level of p-ACCαprotein in the CMV-HGF group was extremely significantly up-regulated(P < 0.01),indicating that HGF is involved in regulating the expression of key genes for adipogenesis.The above results indicate that HGF can alleviate the lipid accumulation of LMH cells induced by high fat by inhibiting the expression of fatty acid production genes.