| Lipid droplets(LDs)are the main subcellular organelles storing lipids in cells,and their core is composed of neutral lipids,with a monolayer phospholipid membrane on the periphery,which plays an important role in regulating the balance of lipids in the body.There are many kinds of proteins distributed on the membrane of lipid droplets,which can synthesize or decompose phospholipids and neutral lipids in lipid droplets,thereby regulating the size and function of lipid droplets.When the CIDE(cell deathinducing DFF45-like effectors)family was first discovered,it was thought to be associated with apoptosis,and named for its high homology to the N-terminal domain of DFF45(DNA fragmentation factor,DFF45).With the continuous research and discovery,it is found that the proteins of CIDE family are localized on the surface of lipid droplets and can form lipid droplet fusion complex(LDFC)at the contact site between lipid droplets and lipid droplets,connect two lipid droplets,promote the fusion between lipid droplets,and thus regulate the size of lipid droplets.It can be seen that CIDEs also have an important regulatory role in the process of lipid metabolism.However,there are few studies on the function of goat CIDEs genes,and the role of CIDEs in the regulation of goat lipid metabolism is still unclear.Therefore,in this study,on the basis of cloning and obtaining the sequences of CIDEs,goat intramuscular adipocytes were used to investigate the regulatory effect of CIDEs on the deposition of goat intramuscular adipocytes using overexpression and RNA interference techniques,and the metabolic pathways of goat intramuscular adipocytes regulated by CIDEs were explored by high-throughput sequencing techniques.These results can provide a theoretical basis for further revealing the role of CIDEs genes in lipid metabolism and their regulatory networks in goats,but also provide a reference for further study of the molecular regulatory mechanism of lipid droplet formation.The main study results are as follows:(1)The sequences of goat CIDEB and CIDEC genes were 923 bp and 1043 bp,encoding 219 and 237 amino acid residues,respectively.(2)CIDEs are widely expressed in goat tissues,among which CIDEA is highly expressed in goat rumen;CIDEB was highly expressed in liver;CIDEC has a high level of expression in the small intestine.There was a very significant positive correlation between CIDEA and the expression of DFFB and ELOVL3 in rumen(P < 0.01),and there was a significant positive correlation with DIO2,TMEM26,PRDM16 and PLIN1(P < 0.05).There was a significant positive correlation between CIDEB and the expression of DFFA and SDR39U1 in liver(P < 0.05).There was a significant positive correlation between CIDEC and the expression of PLIN2,GPLD1 and ADIPOQ in small intestine(P < 0.05).(3)Using eukaryotic expression vector pc DNA3.1.Goat recombinant expression vector was successfully constructed pc DNA3.1-CIDEB and pc DNA3.1-CIDEC,the siRNA of CIDEA,CIDEB and CIDEC genes were successfully screened,and the interference efficiency was 82.1%,82.3% and 88.9% respectively.(4)The lipid droplet accumulation and triglyceride content were increased after overexpression of CIDEA in goat intramyocellular adipocytes,and the number of lipid droplets in adipocytes was decreased and triglyceride content was also significantly decreased after interference with CIDEA.72 differentially expressed genes were screened in the differential gene analysis,and 2 GO lipid metabolism-related pathways and 1 KEGG lipid metabolism-related pathway were obtained by pathway enrichment analysis.RT-q PCR validation results revealed that CIDEA could promote lipid deposition in goat adipocytes by up-regulating the expression levels of genes related to fatty acid and triglyceride synthesis and lipid droplet accumulation and reducing the expression of genes related to lipid degradation.(5)Consistent with the results of CIDEA transfection,overexpression of CIDEB can promote lipid deposition in goat intramuscular adipocytes,interfere with CIDEB and inhibit lipid deposition in goat intramuscular adipocytes.113 differentially expressed genes were found by differential gene analysis.5 GO lipid metabolism pathway and 1 KEGG lipid metabolism pathway was obtained.RT-q PCR results showed that CIDEB could promote lipid deposition in goat adipocytes by up regulating the expression of genes related to fatty acid synthesis and lipid droplet accumulation and reducing the expression level of genes related to lipolysis.(6)The results of CIDEC overexpression and interference were also similar to the transfection results of CIDEA and CIDEB.After overexpression of CIDEC,the lipid droplets and triglycerides in adipocytes increased significantly,while after interference with CIDEC,the lipid droplets and triglycerides in adipocytes decreased significantly.252 differentially expressed genes were found by differential gene analysis.1 KEGG lipid metabolism pathway was obtained.RT-q PCR results showed that CIDEC mainly promoted lipid deposition in goat adipocytes by up regulating the expression of genes related to fatty acid synthesis and lipid droplet accumulation.It was found that SEC14L2,ACOT12,HADHB were important factors in the lipid metabolism regulation network of CIDEs.The above results show that CIDEs can promote the lipid deposition of goat intramuscular precursor adipocytes.SEC14 L2,ACOT12,HADHB are important factors in the lipid metabolism regulation network of CIDEs,which provides reference and theoretical basis for the further study of the regulation network of CIDEs gene in goat lipid metabolism. |
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