| Poultry is an important part of animal husbandry,and the poultry fat traits affect the conversion rate of feed to a certain extent,restricting the development of animal husbandry.Unlike mammals,the lipid metabolism of poultry is mainly occured in liver.Poultry lipid metabolism is regulated by liver related transcription factors,lipid metabolism related genes,microRNA and other factors.VNN1 gene was closely related to lipid metabolism in mammals.VNN1 gene was highly expressed in poultry liver,suggesting that poultry VNN1 gene may play an important role in lipid metabolism.The expression regulatory mechanism of chicken VNN1 gene and its function in the process of liver lipid metabolism was studied in several aspects.The goose VNN1 gene were cloned and its effect on the expression after overfeeding was analyzed.The main results were as follows:(1)The transcription start site of chicken VNN1 gene was determined by 5'RACE method and bioinformatics analysis,locating at 42 bp upstream of VNN1 translation initiation site(chr3:56080855),and the sequence deletion technique showed that the core promoter region of chicken VNN1 was located at-421/-1 upstream of the transcription initiation site.(2)C/EBP? and HNF4? were predicted binding to VNN1 promoter using the bioinformatics software.The relative luciferase of promoter reporter vector significantly redused after C/EBP? and HNF4a mutated(P<0.05),however,over-expression of C/EBP?activated the expression of the VNN1 promoter;the qRT-PCR results showed that the expression of VNN1 mRNA was not significantly changed after over-expression of C/EBP?,but HNF4a up-regulated the expression of VNN1 mRNA(P<0.05),suggesting that VNN1 expression correlated with C/EBP? and HNF4a.(3)251 potential target miRNAs of VNN1 were predicted by using miRNA target prediction software,and the liver metabolism related genes(miR-187-3p,miR-187-5p and miR-33-5p)were selected for further investigation.Dual luciferase reporter assay showed that VNN1 was significantly down-regulated after transfecting miR-33-5p mimic(P<0.01),and the negative effect of miR-3 3-5p on VNN1 was confirmed by the mutation of target site,indicating that miR-33-5p may be the target miRNA of VNN1.(4)Suppressing the expression of VNN1 in primary chicken hepatocytes after treated with siVNN1-1018,and the RNA-seq results showed that 376 genes were differentially expressed,of which 138 were up-regulated and 238 were down-regulated.GO and KEGG analysis showed that there were 25 genes and 16 pathways were involved in chicken lipid metabolism,respectively.Compared with the RNA-seq results,the expression level of FASN,ELOVL2,GDPD5 were significantly up-regulated(P<0.05)and the expression level of E2F7,WNT5B,PLA2G12A(P<0.01)and RACGAP1(P<0.05)were significantly down-regulated,indicating that VNN1 plays an important role by affecting the expression of these genes in chicken liver lipid metabolism.(5)Full-length cDNA of goose VNN1 gene(GenBank accession number:KY399733)was obtained by RT-PCR and RACE,and the full-length cDNA was 1924 bp in length,which contained 1476 bp open reading frame,35 bp 5'UTR and 417 bp 3'UTR,encoding 491 amino acids.After bioinformatics analysis,the VNN1 protein was predicted as a kind of unstable protein with the relative molecular weight of 54870.61 Da and pI of 5.23.The three-dimensional structures of VNN1 protein prediction online between Anser anser and Anas platyrhynchos showed highly similar helical and folding.Sequence analysis showed that the nucleotide and amino acid homology of VNN1 between Anser anser and Anas platyrhynchos were relatively high.Phylogenetic tree analysis showed the close affinity between Anser anser and Anas platyrhynchos.The qRT-PCR results showed that the expression of goose VNN1 gene in the liver was significantly higher than that in the small intestine,kidney and spleen(P<0.01),while the expression of VNN1 gene in goose adipose tissue significantly decreased after overfeeding(P<0.05),suggesting that VNN1 gene may play a role in lipid metabolism. |
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