Study On The Function Of GAS6 Gene In Regulating Adipocyte Differentiation And Its Genetic Effect On Chicken

At present,chicken has become the second-largest meat consumer product in China after pork.With the continuous improvement of the quality of life,high-quality chicken with tender,juicy meat,and unique flavor,gradually become the first choice of consumers.For many years,due to the excessive pursuit of high growth speed of broilers by breeders,modern broiler breeds have experienced excessive deposition of abdominal fat Excessive fat deposition not only wastes diet energy but also affects slaughter performance and reduces production performance of chicken.Therefore,how to solve the problem of excessive deposition of abdominal fat in broiler through breeding is a challenge faced by broiler breeders.Dissection of the genetic regulation mechanism of chicken abdominal fat deposition is an important prerequisite for improving abdominal fat trait using molecular breeding techniques.To explore the genetic regulation mechanism of chicken fat deposition,we successfully isolated chicken preadipocytes and induced differentiation.Then we analysed the gene expression level during the differentiation of chicken preadipocytes using RNA-seq.The results showed that GAS6 gene is significantly up-regulated,indicating its important role in the differentiation of chicken preadipocytes differentiation.This study will analyse the genetic effect of GAS6 on important economic traits of chicken,and verify its function in the differentiation of chicken preadipocytes using molecular biology techniques.The main results are as follows:1.In this study,PCR-SSCP was used to detect single nucleotide polymorphism in GAS6 exon,and correlation analysis was performed with important economic traits of Jinmao Black Chicken The results showed that a C to T mutation(g.C27463T)was detected in exon 10 of GAS6,forming three genotypes of EE,EF and FF.The polymorphism information content,heterozygosity and effective alleles of g.C27463T were 0.352,0.543 and 1.84,respectively.Correlation analysis showed that,in terms of carcass traits,the live weight,carcass weight,semi-eviscerated weight,eviscerated weight,foot weight,breast muscle weight and leg muscle weight of EE genotype individuals were significantly(P<0.05)or very significantly(P<0.01)higher than that of FF genotype individuals.In terms of meat quality traits,the moisture content of breast muscle of FF genotype individuals was significantly higher than that of EF genotype individuals(P<0.05).The crude fat content of EF and FF genotype individuals were very significantly(P<0.01)or significantly(P<0.05)higher than that of EE genotype individuals.The inosine content of EF genotype individuals was significantly higher than that of EE genotype individuals(P<0.05).Therefore,the EE genotype was favourable genotype for chicken carcass traits,and the EF genotype was favourable genotype for the fat and inosinic acid content of leg muscle.In the breeding process of Jinmao Black Chicken,individuals with EE genotype can be selected to improve carcass traits,and individuals with EF genotype can be selected to increase the content of crude fat and inosinic acid in leg muscles,thereby improving meat quality.2.To study the tissue distribution of GAS6 and its developmental expression in abdominal fat of 4 weeks old Jinmao Black Chicken were detected using RT-qPCR technology,and high expression tissues were selected to detect their presence in.The results showed that GAS6 gene was highly expressed in the abdominal fat of male and the female chicken.GAS6 is expressed in the trend of"M" in the abdominal fat of different development stages in male chicken and is expressed in the trend of "W" in female chicken.However,the expression peak of GAS6 gene appeared before 4-week-old both in male and female chicken.These results suggest that GAS6 may participate in the regulation of chicken preadipocytes,thereby affect the early development of abdominal fat.3.The above results have indicated that the polymorphism of GAS6 gene was significantly associated with abdominal fat and intramuscular fat traits of chicken,and GAS6 gene was highly expressed in abdominal fat tissue,suggesting its important role in regulating chicken fat deposition.To verify the function of GAS6 in the differentiation of chicken preadipocytes,Adenovirus interference vectors and overexpressed plasmids were constructed and transfected into chicken preadipocytes.Then we induced preadipocytes differentiation into adipocytes.The expressions of PPARy,C/EBPa and FAS and triglyceride content during differentiation were detected.And the effects of interference and overexpression of GAS6 on the differentiation of chicken preadipocytes were analysed.The results showed that compared with the control group,the expression of PPARy,C/EBPa and FAS in the interference group were inhibited at different stages of differentiation.On the contrary,overexpression of GAS6 promoted the expression of PPAR?,C/EBP? and FAS.Oil red O staining and triglyceride content analysis showed that the content of triglyceride in GAS6 gene interference group was significantly lower than that in the control group(P<0.01),while the content of triglyceride in GAS6 overexpression group was significantly higher than that in the control group(P<0.01).The above results indicate that GAS6 gene promotes the differentiation of chicken preadipocytes.