| Acyl CoA thioesterases(ACOTs)are enzymes that catalyze the hydrolysis of fatty acyl CoA to form free fatty acids(FFA)and coenzyme A(CoA).These enzymes play an important role in lipid metabolism by maintaining the appropriate levels of FFA,fatty acyl CoA and CoA in cells.Acot11,as a member of acots family,is sensitive to the changes of temperature and food intake,so it plays a role in energy storage,inflammation regulation and endoplasmic reticulum stress.As one of the most important aquaculture species in China,yellow catfish has been continuously promoted in new aquaculture technologies,with increasingly diverse breeding methods and significantly improved production.The hybrid yellow catfish was obtained by crossing Pelteobagrus fulvidraco with P.vachelli.The survival rate and disease resistance of hybrid yellow catfish were better than those of other yellow catfish.In this study,acot11β plays an important role in regulating lipid metabolism of hybrid yellow catfish,and this metabolic process is greatly affected by temperature.This study may provide an effective theoretical basis for preventing or reducing metabolic disorder caused by high temperature in yellow catfish.1.Full length transcriptome analysis of hybrid yellow catfishIn this study,we sequenced and established the nanopore sequence library on the platform of nanopore sequence.The liver tissues of hybrid yellow catfish were sequenced and bioinformatics analyzed by using the third generation high-throughput full-length genome sequencing technology.A total of 12.78 GB of clean data was produced by sequencing,and the number of full-length sequences was 11833812.Consensus isoform was obtained by poling the full-length sequences,and 15437 transcripts were obtained by de redundancy of all consensus transcripts.By analyzing the sequence structure of the newly discovered transcripts,7166 complete open reading frame(ORF)sequences were predicted and 8997 new transcripts were annotated.From the perspective of database annotation,Pelteobagrus fulvidraco can be annotated in NR,Swissprot,GO,COG,KOG,Pfam,KEGG databases,but the proportion is relatively small.2.Cloning and analysis of acot11β sequence of hybrid yellow catfishIn this study,rapid amplification of cDNA ends(RACE)was used to clone and bioinformatics analyze the full-length sequence of acot11β in hybrid yellow catfish.The results showed that the full-length sequence was 2452 bp,and the longest ORF was 594 bp.When the gene sequences obtained were compared with the genes in NCBI gene library,A similarity between hybrid yellow catfish and Tachsurus fulvidraco was found to be as high as 94 %,followed by that between hybrid yellow catfish and Pangasianodon hypophthalmus with 76 %.In general,the full-length sequence of acot11β in hybrid yellow catfish is conserved.3.the expression of acot11β sequence and its spatial and temporal expression in hybrid yellow catfishQuantitative real-time PCR(qRT-PCR)was used to detect the expression of acot11β in different embryonic development stages(cleavage stage,blastocyst stage,gastrula stage,neuroembryo stage,tail bud formation stage,adventitia stage,pre larval stage and larval stage)and tissues(blood,skin,liver,brain,muscle,spleen,intestine and kidney).The expression of acot11β was the highest in cleavage stage.With the development of fertilized eggs,the expression of acot11β gradually decreased and then slightly increased during formation of tail teeth.The expression of acot11β in hybrid yellow catfish also showed an upward trend after membrane formation.We speculate that the nutrition sources of yellow catfish are different before and after membrane formation,and the required fatty acid nutrition also changes with the development of embryo.The expression of acot11β was different in different tissues,with the highest in liver,followed by the brain,and the lowest in the blood and spleen.This may mean that acot11β,a gene related to fatty acid metabolism,is more active in tissues where fat storage and fatty acid metabolism take place.4.Effect of temperature on the expression of acot11β in hybrid yellow catfishIn this study,the expression levels of acot11β,acc(acetyl-CoA carboxylase),fas(fatty acid synthase)and pparα(peroxisome proliferator activated receptor α)in hybrid yellow catfish under high temperature stress were determined,and the serum biochemical indexes,liver biochemical indexes and fatty acid contents were also analyzed.The healthy yellow catfish was exposed to 35℃ and compared with the control group(28℃).Compared with the control group,the expression level of acot11β,acc and fas,which controlled lipid synthesis of hybrid yellow catfish was significantly lower than that of the control group,and pparα and other genes promoted lipid decomposition were significantly increased(P < 0.05).So we speculate that high temperature can inhibit lipid synthesis and promote lipid decomposition.The levels of serum glucose(Glu),total cholesterol(TC)and triglyceride(TG)in the experimental group were significantly higher than those in the control group(P < 0.05).Acute heat stress resulted in the decrease of liver glycogen content and an increase of TC and TG content(P < 0.05).We speculate that after the rise of temperature,the glucose in the body is rapidly consumed,which leads to the decomposition of liver glycogen for energy supply,and also leads to the rise of blood glucose;TC and TG are the main storage forms of fat in fish cells,and their rise under high temperature may be due to the down-regulation of gene expression such as acot11β,which makes the fat in fish not easy to absorb,resulting in the disorder of lipid metabolism.At the same time,SFA / MUFA and SFA / UFA were significantly decreased,and FFA of high temperature group was significantly lower than that of control group(P < 0.05).This may indicate that high temperature stress can promote the conversion of saturated fatty acids to unsaturated fatty acids.Meanwhile,high temperature can greatly inhibit the process of FFA lipid synthesis and increase the content of FFA,which means that high temperature may greatly affect the lipid metabolism of the body. |
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