| Adipose tissue is very important in the individual development and plays key roles in the beef quality studies.It has been an important goal to breed cattle with high beef quality which largely depends on the content of the fat in the beef.However,the genes related to lipid accumulation have not been studied widely and large number of genes are needed to uncover because of the complexity of the adipogenesis mechanismsMoreover,alternative splicing can generate multiple transcript products for one gene that increases the mechanism complexity but supplies new study direction as a hot topic in nowadays.Thus,we applied RNA sequencing for subcutaneous adipose tissues from cattle with different ages and genders and analyzed the differentially expressed genes.We screened genes related to adipogenesis in tissue,cell and molecular levels from the differentially expressed genes(DEGs).Finally we studied the tissue specifity of alternative splicing and the influence of alternative splicing to genes related to adipogenesis through silico cloning,constructing tissue profiles of gene expression,analysis the gene expression changing trend during adipocyte differentiation,cellular co-localization,sub-cellular localization analysis and other methods.The main results are as following: 1.RNA sequencing of the bovine subcutaneous adipose tissues and differentially expressed gene statisticsWe applied RNA sequencing for subcutaneous adipose tissues of fetal bovine,adult bulls,adult cows and adult steers,and generated at least 26 million clean reads for each sample.82.58% ~ 84.41% clean reads were successfully mapped on the reference genome.Totally,the expression of 12,233 genes were annotated for further studies.Among the 109 genes commonly and highly expressed(? 500 RPKM)in all the four kinds of adipose tissues,several genes(APOE,GAPDH,FABP4,SPARC,ADIRF and so on)were found related to fat metabolism except 63 genes coding for ribosome proteins.This implied that the 46 non-ribosome related commonly and highly expressed genes could supply candidate genes for adipogenesis study.The expression of genes in adipose tissue of cattle with different genders showed high correlation(r ? 0.91)while only medium correlation(r ? 0.64)were seen for the expression of genes in adipose tissues of cattle in different ages.Combining with the number statistic of DEGs for each comparison pair,we found the influence of age was higher than that of gender in this study.Apart from that,we also detected large number of novel transcript units for each adipose tissue,which will help to improve the annotation of cattle genome and supplied resources for non-coding RNA studies.2.The effects of age and gender to gene expression in adipose tissuesWe detected 2,703 DEGs(Fold change > 2,FDR corrected P < 0.05)through comparing adipose tissue RNA sequencing results between fetal bovine and adult bovine including three replicates(adult bulls,adult cows and adult steers).The GO(gene ontology)terms related to tissue development were significantly(P < 0.05)enriched for GO analysis of the DEGs.We received low p values(slightly higher than 0.05)for the GO terms related to fat metabolism.The core pathway(PPAR pathway)during adipogenesis was significantly enriched and the genes related to lipid accumulation in this pathway were significantly higher expressed in adipose tissues of adult bovine.Moreover,among the 14 marker genes during adipogenesis,12 genes were higher expressed in all adipose tissues of adult bovine than that of fetal bovine.Those proved that the adipose tissue in adult stage was more active than in fetal stage.The GO and pathway analysis for the DEGs among adipose tissues of bovine with different genders showed GO terms related to response to steroid hormone stimulus,fat metabolism and adipokines,and pathways related to adipokines were significantly enriched.This reuslt primarily evidences that the gender will affect the metabolism of bovine subcutaneous adipose tissues and may be related to adipokines.3.Screening genes related to lipid accumulationTo screen genes related to lipid accumulation,we selected 31 candidates genes based on the analysis of RNA sequencing result that including 11 genes expressed in adipose tissues of adult bovine more than 10 times of that in adipose tissues of fetal bovine and 20 genes that commonly and highly expressed in the four adipose tissues.We detected 12 genes(ADIPOR2,CIDEC,GHR,LIPE,S100 B,SCD,THRSP,TUSC5,ANXA2,CST3,SPARC and VIM)that were expressed extremely higher in adipose tissues than in other tissues through constructing expression profiles for different tissues.The expression of 7 genes(GHR,THRSP,SCD,LIPE,TUSC5,CIDEC and CST3)increased during adipogenesis of bovine adipocyte induced by rosiglitazone.We detected 3 genes'(CIDEC,TUSC5 and CST3)expression levels that were significantly increased after over-expressing PPARG2 gene in bovine adipocyte by adenovirus.Finally,we received 3 genes related to adipogenesis and regulated by PPARG2,which added information for the pathway regulated by PPARG2 at the same time.4.Characterization of gene alternative splicing in adipose tissuesTotally,we found 4,753 genes(38.85%)with alternative splicing in the RNA sequencing data of bovine subcutaneous adipose tissues.However,only 1,219(28%)genes alternatively spliced in all the four adipose tissues and only 17% alternative splicing events commonly existed in the four adipose tissues.This illustrated that large differences of alternative splicing events existed among the four adipose tissues.We selected the bovine NFIX gene to further study the tissue specificity alternative splicing.Five different bovine NFIX transcripts were identified by silico cloning and PCR methods.Diverse expression profiles were seen for the five different bovine NFIX transcripts in different tissues of fetal and adult stages.Those studies implied that different isoforms of one gene may have different functions.So it is necessary to study the effects of alternative splicing to genes related to lipid accumulation.5.Alternative splicing model and cellular localization analysis of the adipogenesis genesWe analyzed the alternative splicing model for three genes(CEBPA,CIDEC and TUSC5)related to adipogenesis and compared the cellular localization for the different isoforms of one gene in 293 T or 3T3-L1 cell lines.The two iosoforms of CEBPA were localized in cell nucleus.The two isoforms of CIDEC were localized in cytoplasm and showed no difference.The two isoforms of TUSC5 were localized in cytoplasm but obvious differences were shown.Through analysis of the amino acid sequences for the two TUSC5 isoforms,we found the alternative splicing may affect the cellular localization of the two TUSC5 isoforms by altering the endoplasmic reticulum retention signal.6.The effects of alternative splicing on TUSC5We found differences for the expression profiles of different tissues and the changing trends while adipocyte differentiation between TUSC5 a and TUSC5 b.Moreover,the expression of TUSC5 a was slower than that of TUSC5 b using pDsRed-C1 vectors in 293 T cell lines.Even though they have differences for cell localization and expression,they all localized in endoplasmic reticulum.The CIDEC was not shown localized in endoplasmic reticulum and neither of the TUSC5 isoforms interacted with CIEDC in cytoplasm.In summary,alternative splicing will affect the expression of TUSC5 while has no effect on the interaction between TUSC5 and CIDEC in cytoplasm.Our study constructed transcriptome of Qinchuan cattle subcutaneous adipose tissues,screened the genes related to adipogenesis and further studied those genes in the alternative splicing level.This supplied resources and new clues for further molecular mechanism analyses of adipose tissue development. |
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