The Goose Fat Liver Transcriptome Sequencing At Three Different Stages And The Cloning Of ACAA1 Gene

The liver plays a key role in lipid metabolism,digestion,absorption,synthesis and decomposition.Goose is the best animal model for non-alcoholic fatty liver disease.The goose can deposit a lot of fat in the liver and induce hepatic steatosis by overfeding,but it does not impair the function of the liver.After stopping feeding,the goose liver can be restored to a normal state.The study of goose fatty liver is of great significance in medicine and molecular breeding of livestock and poultry.In this study,the Roche 454 GS FLX sequencing platform was used to sequence liver under three different feeding conditions in Landes geese.The cloned and sequenced ACAA1 gene was analyzed and the results were as follows:1.Through transcriptome sequencing,we obtained a total of 3,824,961 raw reads,consisted of 1,423,917,1,153,743,and 1,247,301 in the NL,FL,and CL group.Including 37,973 isogenes by BLASTX comparison,19,752(52.02%of all sequences)isogenes were found to match the public database.2.Gene functional annotation analysis results:There were 3562 unigenes in GO annotations,1196 in COG annotations,and 285 annotations in KEGG.These genes mainly involve fatty acid synthesis,metabolism,and binding.3.Comparison of differentially expressed gene profiles based on three libraries identified 1,156 differentially expressed transcripts,primarily involving carbohydrate metabolism,lipid synthesis,metabolism,secretion,and storage.We also identified a series of significantly differentially expressed genes related to the biosynthesis and catabolism of fatty acids in goose fatty liver,including LPL,LIPH,HADHA,ACAA,ACOX,and FADS are six candidate genes for adipogenesis that have been screened.4.Verify the expression levels of these genes by qRT-PCR,the results were consistent with the transcriptome data without LIPH gene.The expression levels were significantly elevated in the FL group and significantly down-regulated in CL(P<0.05).5.The full-length cDNA sequence of ACAA1 was cloned by RT-PCR.The result showed that the full-length cDNA was 3,352 bp,including 1,323 bp in coding sequence,77 bp in 3'UTR,and 1,952 bp in 5'UTR.6.Bioinformatics analysis showed that the molecular weight of ACAA1 protein in Landes goose was 45.0 kDa and the isoelectric point was 9.19.The protein contained 19 kinds of amino acids,which the highest glycine and alanine content was 14 3%and 12.7%,respectively.7.Liver tissue sections revealed that fatty liver deposited large amounts of lipid droplets during overfeding.Among this period,the expression level of ACAA1 gene was detected and showed increasing firstly,then decreasing.The ACAA1 expression was the highest at 14 d by overfeding,followed by a rapid decline.At the sametime,the expression in the overfeding group was significantly higher than that in the control group(P<0.01).In summary,six differentially expressed genes related to adipogenesis and metabolism were screened by transcriptome sequencing,and the full-length ACAA1 gene sequence was cloned.In addition,it was found that overfeding can significantly promote the ACAA1 expression.This provides a theoretical basis for the molecular breeding of geese and the study of subsequent functions.