| Goose is a herbivorous poultry and popular among consumers.The World Health Organization has listed goose meat at the top of the "Healthy Food".As we all know,geese are waterfowl and have a strong ability to deposit fat,especially in the abdomen and subcutaneous fat.Excessive deposition of fat reduces the feed conversion rate of the goose,reduces the carcass yield,and affects the reproductive system function.Therefore,how to reduce abdominal and subcutaneous fat deposition has become one of the urgent problems to be solved in the sustainable development of the industry.However,the deposition of fat in different muscular tissues has a certain correlation,that is,when reducing the deposition of abdominal and subcutaneous fat,the deposition of intramuscular fat also reduces,which in turn results in a negative impact on the flavor and quality of goose meat.Therefore,in order to reduce abdominal and subcutaneous fat deposition without damaging meat quality,the differences in physicochemical properties and regulatory mechanisms of fat deposition in different muscular tissues needs to be characterized.Therefore,we firstly compare the histological and physiological characteristics of abdominal,subcutaneous and intramuscular fat in Yangzhou geese at embryonic,early growth,and development stages,respectively.Candidate genes for the specific differentiation of adipose precursor cells was then determined.Finally,the role and its molecular mechanism of these candidate genes in the specific regulation of adipogenic differentiation were verified.Our study may identify the molecules of differential deposition of fat in the intramuscular,subcutaneous and abdominal tissue and will provide new target genes for reducing abdominal and subcutaneous fat deposition or increasing intramuscular fat deposition in goose.The main contents are as follows:1.The rules of intramuscular,abdominal and subcutaneous fat deposition in different developmental stages of geeseIn order to explore the developmental rules,histological characteristics and physiological characteristics of intramuscular fat,subcutaneous fat and abdominal fat tissue at different growth stages,HE staining and RT-qPCR were adopted to analyze the morphology characteristics and cytological parameters of fat tissues.The results showed that subcutaneous adipose tissue was the main area of fat deposition during embryonic stage and developed earlier than abdominal fat.After birth,the deposition of abdominal fat accelerated,and the relative weight of abdominal adipose tissue was significantly higher than that of subcutaneous adipose tissue at 120 days(P<0.05).During the whole period,no obvious intramuscular fat deposition was observed.The fat was mainly deposited in the form of cell hypertrophy,and the volume of subcutaneous and abdominal fat cells increased significantly in embryonic stage(P<0.05).After birth,the subcutaneous fat cells still increased significantly(P<0.05)while the abdominal adipocytes remained stable(P<0.05).The diameter and cross-sectional area of abdominal adipocytes were significantly larger than those of subcutaneous adipocytes at 120 days(P<0.05);during the whole developmental stage,the TG content in the pectoral muscle was generally at a low level,and the TG content in the abdominal adipose tissue was higher than that in the subcutaneous adipose tissue after 28 days.The RT-qPCR result showed that the expressions of lipid uptake and lipogenesis-related genes Lpl,aP2,Ppara,etc.in abdominal and subcutaneous adipose tissue,showed a trend of increasing at early stages and then decreasing thereafter,while the expressions of decomposition-related genes Atgl in intramuscular and subcutaneous adipose tissue showed a increase and then a decrease trend.Finally,the differences in the proliferation and differentiation abilities of the three types of preadipocytes were compared,and it was found that the growth rate of abdominal and subcutaneous preadipocytes was faster than that of intramuscular preadipocytes.During induction and differentiation,TG deposition was the highest in subcutaneous adipocytes and the lowest in intramuscular adipocytes.Adipogenic differentiation-related genes Ppary have different expression patterns during the differentiation of subcutaneous,intramuscular and abdominal preadipocytes.The above results showed that there were significant differences in the developmental capacity,adipocyte expansion capacity,lipid deposition capacity,lipid metabolism capacity,and the proliferation and differentiation capacity of intramuscular,abdominal and subcutaneous fat.2.Screening differentially expressed genes during adipogenic differentiation in abdominal,intramuscular and subcutaneous adipose precursor cellsIn order to identify the specific regulatory targets of abdominal,subcutaneous and intramuscular fat deposition in goose,high-throughput transcriptome sequencing was performed in the constructed intramuscular,abdominal and subcutaneous preadipocyte groups.The results showed that after differentiation induction,a total of 4037,3330 and 3161 differentially expressed genes were screened in the adipogenic different iation of subcutaneous,intramuscular and abdominal preadipocytes,respectively.The KEGG Pathway analysis showed that differentially expressed genes were enriched in adipocyte proliferation and differentiation related pathways,such as the interaction between extracellular matrix components(ECM)and receptors,the insulin signaling pathway PI3K-Akt,MAPK and et al.Subsequently,we screened out the important regulators in intramuscular,subcutaneous and abdominal fat differentiation,such as Ppary,LOC106044608,Acap3,Sox4,LOC106043511 and others that were differentially expressed during the adipogenic differentiation of subcutaneous and abdominal preadipocytes,while no differences in intramuscular preadipocytes.In addition,other important regulators,such as Bmp4,Pls3,Igfbp2,Klf15,Klf12,were differentially expressed during adipogenic differentiation of intramuscular,subcutaneous and abdominal preadipocytes.Therefore,the differential genes such as Ppary and Bmp4 are selected as candidate targets for further studies in differential deposition of subcutaneous,abdominal and intramuscular fat.3.Effects and molecular mechanisms of Ppary on intramuscular,abdominal and subcutaneous fat depositionIn order to elucidate the specific mechanism of Ppary in intramuscular,abdominal and subcutaneous fat deposition,the complete coding region sequence of Ppary was obtained.In addition,the expression of Ppary gene in abdominal fat and sebum was much higher than that in other tissues(P<0.05),and its expression levels were in the order of abdominal fat>sebum>pectoralis muscle>kidney>liver>lung>spleen>heart>muscle>stomach.The Ppary overexpression vector and siRNA were further transfected into the three types of preadipocytes,respectively.The overexpression of Ppary significantly increased the deposition of TG in adipocytes(P<0.01).The interference of Ppary by siRNA could significantly reduce the content of TG in abdominal adipocytes(P<0.05),but no significant effects in intramuscular and subcutaneous adipocytes(P>0.05).Furthermore,Ppary inhibitor T0070907(0 ng/mL,12.5 ng/mL,25 ng/mL,50 ng/mL)was added to the three types of preadipocytes.The 25 ng/mL T0070907 could significantly inhibit the TG production in abdominal adipocytes(P<0.05),but no effects on TG production in intramuscular and subcutaneous adipocytes(P>0.05).Our results revealed the specific mechanism of Ppary in the intramuscular,abdomen and subcutaneous fat deposition,and provided a new target for reducing abdominal fat deposition and improving feed conversion rate in geese.4.Effects and molecular mechanisms of Bmp4 on intramuscular,abdominal and subcutaneous fat depositionIn order to elucidate the specific mechanism of Bmp4 in intramuscular,abdominal and subcutaneous fat deposition,the complete coding region sequence of Bmp4 was obtained.In addition,the expression of Bmp4 in breast muscle,abdomen and subcutaneous fat was much higher than that in other tissues(P<0.05),and its expression levels were in the order of breast muscle>abdominal fat>sebum>liver>kidney>stomach>lung>spleen>heart.During the differentiation of abdominal and subcutaneous preadipocytes,the expression of Bmp4 was significantly higher at 0 h than 72 h and 144 h of differentiation(P<0.05).During the differentiation of intramuscular preadipocytes,the expression of Bmp4 at 72 h was significantly lower than 0 h and 144 h of differentiation(P<0.05).The Bmp4 overexpression vector and siRNA was further transfected into abdominal,intramuscular and subcutaneous preadipocytes,respectively.The overexpression of Bmp4 significantly increased the deposition of TG in adipocytes(P<0.05).The interference of Bmp4 by siRNA significantly reduced the content of TG in abdominal adipocytes(P<0.05),but had no significant effects in intramuscular and subcutaneous adipocytes(P>0.05).Furthermore,Bmp4 activator SB4(0,25,50,100 ng/mL)and inhibitor LDN193189(0,12.5,25,50 ng/mL)were added to the three types of preadipocytes.We found that 25 ng/mL Bmp4 activator SB4 treatment could significantly increase the deposition of TG in intramuscular adipocytes(P<0.05),but no significance in abdominal and subcutaneous adipocytes(P>0.05).Our results revealed the specific mechanism of Bmp4 in the abdominal and subcutaneous fat deposition of goose,and provided a new target for increasing intramuscular fat deposition and improving meat quality in goose.In conclusion,there were significant differences in the developmental capacity,adipocyte expansion capacity,lipid deposition capacity,lipid metabolism capacity,and the proliferation and differentiation capacity of intramuscular,abdominal and subcutaneous fat.High-throughput transcriptome sequencing revealed the overall regulatory network of intramuscular,subcutaneous and abdominal adipocyte differentiation at the molecular level,and screened out Ppary and Bmp4 genes as candidate targets for further research on differential deposition of subcutaneous,abdominal and intramuscular fat.Further,the specific interfering siRNA and overexpression vector of Ppary and Bmp4 were constructed,and Ppary and Bmp4 regulate the differential deposition of abdominal,intramuscular and subcutaneous fat deposition by altering the expression levels of fat synthesis and decomposition genes,respectively.In addition,25 ng/mL Ppary inhibitor T0070907 can significantly inhibit the deposition of TG in abdominal adipocytes,and 25 ng/mL Bmp4 activator SB4 can significantly increase the deposition of TG in abdominal adipocytes.Our research may provide important clues for reducing abdominal fat deposition or increasing intramuscular fat deposition in goose. |
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