| Polyunsaturated fatty acids(PUFAs)are important forms of fatty acids in the animal’s body.They play an important biological role by participating in the body’s lipid metabolism,growth and development,immune function,cell membrane function,and cardiovascular disease.Fatty acid desaturases(FADS)are key enzymes in the biosynthetic pathway of PUFAs,and their expression is affected by many factors.However,in poultry(chicken),there are few reports about the expression regulation and action mechanism of the FADS gene family.In this study,a variety of bioinformatics analysis software was used to systematically analyze the collinear relationship,genetic evolution relationship,gene structure,conserved motifs and functional domains of chicken FADS gene family members;Real-time quantitative PCR(real-time quantitative PCR,qRT-PCR)technology was used to analyze the expression of FADS gene family members in different tissues of 30-week-old chickens and chicken liver tissues at different physiological periods;Study the effect of estrogen on the gene expression of FADS family members through in vivo experiments;The dual luciferase reporter system was used in combination with overexprcssion and interference experiments to identify the transcriptional expression regulators of FADS2 gene;Online software analysis was used to analyze microRNAs(miRNAs)potentially associated with the 3’ untranslated regions(3’ UTR)of the FADS2 gene and the miRNA mimics transfection test to explore the regulation of the FADS2 gene by miRNAs from the post-transcriptional level.The main findings are as follows:1.The results of biological characteristics analysis showed that in the colinear analysis of the FADS family,the FADS3 gene of poultry might not exist or has not yet been annotated or lost during evolution,the FADS1L1 and FADS1L2 genes exist only in chicken,and the chromosome segment of the FADS6 gene was more conserved among species;The results of phylogenetic tree analysis of the FADS family showed that the FADS6 gene was clustered separately among species,and other members of the FADS gene family were clustered;The FADS6 gene differs greatly from other members in the number of exons and the composition of conserved motifs among species;The results of protein functional domain prediction analysis showed that except FADS6,the same functional domain(Cyt-b5)exists in the proteins encoded by chicken FADS],FADS2,FADS1L1 and FADS1L2 genes,suggesting that the FADS6 gene might be functionally different from other members of the FADS family.2.The results of spatiotemporal expression analysis of FADS family tissues showed that all members of the FADS family were expressed in the heart,liver,spleen,lung,kidney,duodenum,pancreas,glandular stomach,and pectoral muscle tissue of 30-week-old Lushi green-shell hens,of which FADS1,FADS2 and FADS6 had the highest relative expression level in the liver(P<0.05),FADS1L1 had the highest relative expression level in the pectoral muscle,and FADS1L2 had the highest relative expression level in the pancreas(P<0.05);The expression analysis results of the FADS family in chicken liver at different periods showed that the expression levels of each member of the FADS family in the liver of hens at 30 weeks were significantly higher than those at 20 weeks(P<0.05).3.The results of estrogen-treated 10-week-old Lushi green-shell laying hens showed that compared with the control group,estrogen(0.5 mg/kg)could significantly increase the expression of FADS1,FADS2 and FADS1L1 genes in chicken liver(P<0.05),had no significant effect on the expression of FADS6 and FADS1L2 genes(P>0.05).4.Through bioinformatics prediction analysis,it was found that CEBPβ and SREBP are two potential transcription factors in the core promoter region of FADS2 gene;Analysis of overexpression and interference experiments on LMH cells revealed that overexpression and interference of CEBPβ had no significant effect on the expression of FADS2 gene(P>0.05);Interfering with SREBP1 could significantly down-regulated the expression of FADS2 gene(P<0.05),and interfering with SREBP2 gene had no significant effect on the expression of FADS2 gene(P>0.05).It was further verified at the protein level that SREBP1 could significantly affect the expression level of FADS2 gene(P<0.05);E-box-Like SRE wild type or mutant recombination vector with SREBP1 binding site,co-transfected with chicken LMH cell line with PGL4.73(carrying Renilla luciferase gene)vector.The results showed that the fluorescence activity ratio of E-box-Like SRE mutant carrier was significantly lower than that of wild-type carrier(P<0.05),which proved that SREBP1 regulated the transcription of FADS2 gene by combining the E-box-Like SRE element in the core region of FADS2 gene promoter,and finally affects the level of FADS2 protein.Moreover,the expression level of SREBP1 in chicken liver at 30 weeks of age is significantly higher than that at 20 weeks of age(P<0.05),which again proves that SREBP1 is a transcription factor of FADS2 gene,due to the change in the expression level of transcription factors,the expression level of FADS2 gene at 30 weeks old was significantly up-regulated compared to the change at 20 weeks old.In addition,the results of PPARs agonist treatment of LMH cells showed that both PPARa and PPARy could significantly increase the expression level of FADS2 gene(P<0.05).5.The experimental results of candidate miRNAs overexpression in the 3’UTR of the FADS2 gene showed that overexpression candidates miR-30c-1-3p,miR-6665-5p,and miR-6608-3p all had no signi ficant effect on FADS2 gene expression(P>0.05).In summary,the poultry FADS3 gene might not exist or had not been annotated or lost during evolution,and the FADS1L1 and FADS1L2 genes were only present in chickens,the FADS6 gene was significantly different from the FADS1,FADS2,FADS1L1 and FADS1L2 genes in phylogenetic tree,gene structure,conserved motif analysis and protein functional domain prediction.The FADS gene family was widely expressed in chicken tissues,and higher abundance in liver and pancreas relative to the other tissue detected;The relative expression of FADS gene family at the peak of egg production(30 weeks of age)was significantly higher than that of pre-laying(20 weeks of age).Estrogen promoted FADS1,FADS2 and FADS1L1 gene expression,but did not affect FADS6 and FADS1L2 expression.At the genome level,SREBP1 was a transcription factor that initiated FADS2 gene expression;at the post-transcription level,FADS2 gene expression was not regulated by miR-30c-1-3p,miR-6665-5p,and miR-6608-3p.PPARa and PPARy could increase the expression level of FADS2 gene.The results of this study provided the basis for further research on the action mechanism of the FADS gene family. |
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