| The necessary guarantee for the sustainable development of the contemporary aquaculture industry is high-quality and high-energy feed.Appropriate feed lipid levels can promote the growth of farmed fish,but the ability of fish to adapt to lipid and the adaptation mechanism are not yet clear.In this study,Micropterus salmoides was taken as the research object.From the analysis of liver transcriptomics after feeding high-lipid diet to Micropterus salmoides,the key research object,the key regulator of fatty acid oxidation—carnitine palmitoyl transferase I?cpt1?—was identified.To further explore the expression difference of cpt1 gene in Micropterus salmoides tissue and its response to lipid level and growth stage.The research results can analyze the nutrition and physiological adaptation mechanism of Micropterus salmoides to high-lipid feed from a molecular level,and provide a theoretical basis for the development of high-quality and high-energy feed with optimal lipid levels.The main research results and conclusions are as follows:1.Liver transcriptome and intestinal microbe analysis of Micropterus salmoides after high-lipid diet and cpt1 gene cloningThe purpose of this study was to find important regulatory genes for lipid factors through high-throughput sequencing and explore the nutritional and physiological adaptation mechanism of Micropterus salmoides to high-lipid diet.A total of 3 treatment groups were set in the study,with 3 repetitions in each group and 25 fishes per repetition.Prepare three lipid-level iso-nitrogen experiment diets with low lipid?6%?,medium lipid?12%?and high lipid?18%?.The initial weight of Micropterus salmoides is?23.6 ± 1.0?g,and the experimental period is 10 weeks.After extracting Micropterus salmoides liver m RNA,sequencing and filtering low-quality data,a total of 315,645,776 clean reads were obtained.Trinity software was used for splicing and screening,and a total of 130613 Unigenes were obtained.Among them,65,683 Unigenes were successfully annotated in 7 public databases,accounting for 50.28% of the total number of Unigenes.There also have 30.25% of Unigene annotated in the NR database,and 43.5% is annotated in the NT database.According to differential expression analysis?fpkm>0.3?,compared with the lower lipid group,there were 761 differential genes in the high-lipid group,of which 289 differential genes were up-regulated and 472 differential genes were down-regulated.According to KEGG-pathway enrichment analysis,the differentially expressed genes are mainly enriched in 6 lipid metabolism pathways such as the adipokine signaling pathway,AMPK signaling pathway,PPAR signaling pathway,lipolysis regulation in fat cells,fat digestion and absorption,and biosynthesis of unsaturated fatty acids?padj<0.05?,etc.Similarly,most genes encoded by the intestinal flora of Micropterus salmoides in the high-lipid group were significantly enriched in lipid transport and metabolic pathways in COG functional annotation and KEGG metabolic pathway analysis.Through the analysis of lipid metabolism related pathways,cpt1 was finally selected as the main research gene for lipid metabolism in this study.Using RT-PCR and SMART RACE technology,the full-length sequence of cpt1 gene was cloned.The results showed that the cpt1 sequence of Micropterus salmoides includes a complete open reading frame of 1251 bp and encodes a protein consisting of 417 amino acids.According to the amino acid sequence analysis,the relative molecular mass of cpt1 is 47.3 k D,the theoretical isoelectric point is 5.78,the lipid index is 80.26,and its hydrophilic index is-0.405.It is mainly located in the cytoplasm and has no transmembrane structure area.Protein structure prediction revealed that there were 28 Ser phosphorylation sites,19 Thr and 14 Tyr phosphorylation sites in Micropterus salmoides cpt1;the protein instability coefficient is 42.56,which is an unstable protein,and this may be related to its characteristics as an enzyme involved in catalytic reactions.Through sequence blast identification,Clustalw X multiple sequence alignment and phylogenetic tree analysis,it is known that the cpt1 gene of the Micropterus salmoides is highly conserved.This data provides a valuable theoretical basis for studying the regulatory mechanism of the Micropterus salmoides cpt1 gene from the perspective of signaling pathways and molecular biology.2.Differences in cpt1 expression and the response patterns in Micropterus salmoidesIn order to explore the effect of sampling time on the expression of cpt1 in Micropterus salmoides,determine the appropriate sampling time,and explore the differences of cpt1 expression in tissues of Micropterus salmoides and the effects of different lipid levels and different growth stages on gene expression at the molecular floor.1.In this experiment,the Micropterus salmoides were fed after 48 hours fasting,and liver samples were taken at 0h,1h,2h,4h,6h,9h,12 h,24h,36 h,and 48 h,respectively.Three fishes were taken at per time point,and three samples of each fish were taken at a fixed site in the liver as a biological replicate to do the Real-time fluorescence quantitative experiments.And the relative expression data of cpt1 gene were obtained using the 2-??ct method.The results showed that the expression of cpt1 in the liver of Micropterus salmoides was highest at 1-2h after feeding?P<0.05?;the expression at 0h,9h,12 h and 48 h after feeding was followed;and the expression of cpt1 was lower at 24 h and 36 h after feeding;while the expression is lowest at 4-6h after feeding?P<0.05?.This revealed that the sampling time had a significant effect on the expression of cpt1 gene?P<0.05?,and 1-2h after feeding is the best sampling time to study the expression of cpt1 gene in the liver of Micropterus salmoides.2.During the stage sampling,1h after feeding the Micropterus salmoides,9 tissues including liver,foregut,kidney,spleen,stomach,muscle,gill filament,brain and heart of Micropterus salmoides in low-lipid group,medium-lipid group and high-lipid group were taken and subjected to q PCR experiments.The data showed that the cpt1 gene was highest expressed in liver and foregut,followed by kidney and spleen,and lower in stomach,brain,heart,gill filament and muscle?P<0.05?.3.Under different lipid levels,the expression of cpt1 was highest in the tissues of the medium-lipid group?12%?,followed by the high-lipid group?18%?,and the lowest in the low-lipid group?6%??P<0.05?.4.Tissue samples of Micropterus salmoides were taken on the 30 th,60th and 90 th days of breeding,respectively,and subjected to q PCR experiments.The results showed that the expression of cpt1 in different growth stages increased with the lengthening of feeding time.The above results indicate that feed lipid level and feeding time can induce the expression of cpt1 gene in Micropterus salmoides,and cpt1 may be involved in the catabolism of body lipid.This data can deeply explore the physiological regulation mechanism of Micropterus salmoides on high-lipid feed,and it can also improve the theoretical reference for the development of high-quality and high-energy diet formulated with optimal lipid levels. |
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