Molecular Cloning,Characterization And Functional Identification Of SLC35D3 Gene

The deposition of fat in pigs affects the production efficiency,meat quality and consumer's choice of meat.Among them,the content of intramuscular fat is one of the main factors affecting meat quality,directly affects the flavor,tenderness and color index.SAT and lean meat percentage has a negative correlation,that directly relates to the economic value and meat quality.Therefore,the key issue is how to reduce the amount of SAT deposition meanwhile increase the content of intramuscular fat.Visceral fat,such as PAT,however,has a high metabolic activity,and is more sensitive to insulin resistance and adrenergic stimulation,so visceral fat has better predictive function.Studies have shown that the SLC35D3 gene is associated with obesity and metabolic disorders,however the function of SLC35D3 need further research.In this study,the obese Meishan pigs,mini-obese Bama pigs,and the lean type Yorkshire which has low intramuscular fat content were selected as experimental animal model.The cDNA sequence of SLC35D3 gene was obtained by RACE technology.Using RT-qPCR the SLC35D3 gene expression level was obtained.Our results showed that the gene was highly expressed in adipose tissue and muscle tissue.Further we choose LD,SAT,PAT and LL of three breeds pigs for specific study.It was found that there was a negative correlation between the intramuscular fat content and the body fat percentage of three breeds of pigs,which indicated that the expression of SLC35D3 may be closely related to the adipose deposition in pigs.Finally,the RNAi technique was used to further identify the underlying mechanism of SLC35D3 gene in the regulation of adipose deposition at the transcriptional level.(1)Cloning and sequence analysis of SLC35D3 geneFirst of all,the cDNA sequence was cloned through the RACE technology,the obtained length cDNA of 2238 bp contained a 1272 bp ORF and 966 bp 3' UTR.The ORF encodes a polypeptide of 423 amino acids;BLAST results showed that the amino acid sequence of pig SLC35D3 had the closest phylogenetic relationship with the ovis,it is slightly different from the human,gorilla,and mouse,but still belongs to the same large branch.Further II bioinformatics analysis showed that SLC35D3 protein contains 8 transmembrane domains,there is no signal peptide structure.The secondary structure of SLC35D3 protein mainly contained a-helix(38.71%)and random coil(36.41%).The hydrophobic region is characterized by alpha helical structure.The functional site contains 3 O-glycosylation sites,42 phosphorylation sites,without N-glycosylation sites.Clone and sequence analysis of SLC35D3 gene provide a foundation for the further study of SLC35D3 gene.(2)The differences expression pattern of SLC35D3 during different tissues and breedsThe mRNA level detection of SLC35D3 showed that SLC35D3 gene was highly expressed in LD and adipose tissue,and low in other tissues.These results suggest that SLC35D3 may be involved in the regulation of fat metabolism.Expression patterns among different breeds showed that,in the SAT and PAT,SLC35D3 gene expression level in Meishan Bama pigs is lower than Yorkshire,and there was significant difference in PAT(P<0.05).In LL and LD,the SLC35D3 gene expression was the highest in Yorkshire,followed by Meishan and Bama pigs,the differences were significant(P<0.05).The level of protein was detected by western blot,and the results were consistent with mRNA expression level.These results showed that there was a negative correlation between the intramuscular fat content and the body fat percentage of three breeds of pigs,which indicated that the expression of SLC35D3 was closely related to the fat deposition of pigs.(3)The effect of SLC35D3 gene silencing on intramuscular adipocyte differentiationIn this study,we first isolated and cultured porcine intramuscular preadipocytes,and then using RNAi technology to study the molecular mechanism of SLC35D3 gene on fat deposition.The effective interference fragment of siRNA-3 SLC35D3 was selected,the interference effect was 50%.After SLC.35D3-siRNA was transfected into intramuscular preadipocytes,compared with the negative control group,expression level of adipocyte differentiation marker gene such as PPARy,was increased significantly(P<0.05),expression level of lipogenesis key genes including aP2 and SREBP-1c was also significantly increased(P<0.05),the expression level of C/EBPa was increased but not significantly.At the same time,the expression level of ATGL(P<0.05),HSL and Sirtl(P<0.05)was decreased.In addition,the content of FFA in the medium was significantly increased(P<0.001),but the lipid droplets were increased.The results showed that the expression level of genes related to the synthesis and metabolism of adipocytes was increased after the expression of SLC35D3 gene was inhibited,while the expression of the gene involved in Catabolic reaction was decreased,and the deposition of intramuscular fat was significantly increased.In brief,the SLC35D3 gene negatively regulates the expression of genes involved in the synthesis of intramuscular fat cells,and inhibits the differentiation of adipocytes,reduces the amount of fat synthesis,to affects the deposition of intramuscular fat.This study successfully cloned SLC35D3 gene,and a series of bioinformatics analysis was carried out.The Meishan pig tissue expression analysis showed that,the SLC35D3 gene is highly expressed in adipose tissue and LD tissue.The expression level in adipose tissue and LD tissue with three breeds showed that the SLC35D3 was negatively correlated with intramuscular fat content and body fat rate changes.Last,the RNAi technology was used at the cellular level to further verify the negative regulation of fat deposition of SLC35D3 gene.