| The fat-tailed sheep was originally evolved from wild ancestor of thin-tailed sheep,experiencing a long period natural selection and artificial breeding.Up to the present,a considerable amount of the fat-tailed sheep population has been formed,accounting for 25%of the world's sheep population.In China,there are more abundant fat-tailed sheep breeds,and their tail fats were once considered to be desirable food sources.However,with the alteration of eating habits in consumers in recent years,fat tail has become one of the factors restricting the development of modern sheep industry.Therefore,by intervention of body fat distribution in fat-tail sheep,the fat tail could genetically convert to a thin tail so as to lower the total fat of carcass and enhance the economic value of sheep.In this study,we compared the differences in fat distribution pattern between fat-tailed sheep and thin-tailed sheep,identifying many candidate genes associated with the depot-specific regulation and fat deposition in sheep tail region,accompanied by function study of key gene in primary adipocyte isolated from tail fat in sheep through gene editing techniques.These findings provided the molecular basis for the selection of advantage trait in sheep.The main results in our study are as follows:1.Preparing the paraffin section,determining fatty acid composition and surving gene expression of SAT,VAT and TAT from different anatomical locations,we found that fat cell volumes of Tibetan sheep were significantly larger than those of Tan Sheep with the same age.The SAT and TAT cells were significantly larger than the VAT cells.Among the eleven fatty acids determined,the saturated fatty acids?SFA?and monounsaturated fatty acids?MUFA?accounted for more than 90%of the total fatty acid content,the remaining was the polyunsaturated fatty acid?PUFA?.C16:0 and C18:0 are the two main types of fatty acids in SFA and C18:1n-9 has the the highest concentration in MUFA.Regardless of Tan Sheep or Tibetan Sheep,the SFA contents in SAT and TAT were lower than in VAT,while the MUFA contents were higher than in VAT.SCD1 and FASN,as two key genes influencing fatty acid synthesis,exhibited highly expression in adipose tissues of Tan Sheep and Tibetan Sheep,displaying the expression trend of“high-low-high”in SAT,TAT and VAT.2.Using the RNA-Seq technique,we profiled the transcriptome of TSATs,TVATs,TTATs,OSATs,OVATs and OVATs from Tan Sheep and Tibetan Sheep and constructed a total of 18 cDNA sequencing libraries.Totally,1,058 DEGs were identified among three adipose depots in Tan Sheep when we performed differential expression gene?DEGs?analysis,of which TVAT vs.TTAT had the highest number of DEGs.It was worth emphasizing that a set of Homeobox genes exhibited depot-specific expression manner.The results of gene expression clustering and principal component analysis showed the gene expression profile of TTAT was distinct from those of other two adipose depots?TSAT and TVAT?.Based on the similar gene expression pattern,we conducted the analysis of the clustering and found 196,531 and 331 up-regulated DEGs in TSAT,TVAT and TTAT,respectively.Many genes from TVAT are involved in some biological pathways related to inflammatory response,immune defense and infectious diseases.In three adipose depots of Tibetan Sheep,we totally identified 711 DEGs,of which OSAT/OTAT,OVAT/OTAT and OSAT/OTAT had the number of 522,441 and 0 DEGs,respectively,suggesting OSAT and OTAT are the similar type of fat tissue,while they are different with OVAT.There may be different in the origin of adipocyte progenitor cells.711DEGs were divided into two groups using the sub-cluster analysis:one was that the DEGs in OSAT and OTAT expressed at higher levels than in OVAT;the other was that the DEGs in OVAT exhibited higher expression abundance than in OSAT and OTAT.These genes may play a regulatory role in the development and maintenance of corresponding adipose tissue.We further analyzed the GO function enrichment and KEGG pathway of DEGs in each sub-cluster and found that some DEGs which expressed highly in OSAT and OTAT were mainly participated in the pathways including“Fatty acid synthesis and metabolism”,“Adipocyte differentiation”,“Carbohydrate metabolism”and so on.However,the genes with the highest expression in OVAT were associated with many biological progresses,such as immune system,inflammation,cell adhesion molecules,cytokine-cytokine receptor interaction and diseases.In addition,there were some DEGs that expressed uniquely in OVAT,such as ALDH1A3,TCF21,RDH16 and UCP1 which were linked to nutrient metabolism and energy balance.3.The study of tissue expression profile showed that ALDH1As expressed widely in the various tissues of Tan Sheep and Tibetan Sheep,displaying predominately expression tendency of liver,lungs,kidneys and visceral fat,while lower or undetectable levels in rumen,muscle and skin.Combined with FPKM value from RNA-Seq data,we found that the expression of ALDH1A1 in adipose tissues were significantly higher than those of ALDH1A2and ALDH1A3.Subsequently,we isolated preadipocyte from tail fat in Tan Sheep,and detected the locations of ALDH1As in these cells using the method of immunofluorescence stain,finding that ALDH1As are mainly located in the cytoplasm.During the differentiation of preadipocyte into adipocyte,ALDH1As expression levels were examined by the methods of q-PCR and Western blot.The results indicated that the expression of ALDH1A1 has been greatly improved after stimulating preadipocytes by MDI medium.However,ALDH1A3 was not significantly changed before and after adipocyte differentiation.Unexpectedly,ALDH1A2was not detectable.These results suggested that ALDH1A1 may regulate adipocyte differentiation while ALDH1A2 and ALDH1A3 may have compensating effect only when cells were under certain circumstances.4.The coding sequence of ALDH1A1 gene was firstly cloned from fat tissues of the subjects,and then was inserted into recombinant adenoviral expressing vector,and successfully packaged Ad-ALDH1A1 with 293A packaging system.Using the CRISPR/Cas9gene editing system,ALDH1A1 gene was genetically modified by genetic deletion mutation,and ALDH1A1 defective precursor cell model was successfully constructed.We analyzed the knockout efficiency of each pair of sgRNA by the assays of T7E1 enzyme digestion,reaching up to 65.16%.Moreover,the types of mutations were diverse,including single or multiple base mutations,deletions and insertions,mostly occurring near the target sequence,also at the distance of 200 bp from the target site,even deleting or inserting the larger DNA fragments.The positive cells produced by sgRNA4 with the highest cutting efficiency were selected as the seed cells to screening monoclonal cell,proliferating and obtaining a double-knocked hybrid ALDH1A1-/-preadipocyte.The transcription activity of ALDH1A1 in ALDH1A1-/-preadipocyte was greatly reduced,and the corresponding protein expression decreased significantly.Most importantly,mRNA levels of some genes related to fat cell differentiation were strikingly down-regulated,including PPAR?,FASN,SCD1,LPL,ADIPOQ and LEP,and the accumulation of lipid droplets decreased when compared to wild-type preadipocyte,suggesting that the ALDH1A1 deficiency in preadipocyte suppressed adipicyte differentiation.The overexpression of ALDH1A1 in WT preadipocyte reduced LPL and FASN mRNA levels;However,the ectopic expression of ALDH1A1 in ALDH1A1-/-preadipocyte increased the expression of C/EBP?,may partly rescue the differentiation capacity of ALDH1A1-/-cells.In conclusion,the diameter of the fat cells in TAT,SAT and VAT from Tan Sheep and Tibetan Sheep were decreased in turn.The SFA in SAT and TAT were lower than VAT while the MUFA in SAT and TAT were higher than in VAT.There are the greatest transcripotome differences between TVAT and TTAT in Tan Sheep,however,OVAT was distincted from OSAT and OTAT.Moreover,ALDH1A3,UCP1 and HOXC13 may be as the potential candidate genes for the distribution and deposition of adipose tissues.ALDH1A1,CYP26B1and CYP26C1 may participate in the regulation of adipocyte differentiation in Tan Sheep.The deletion of ALDH1A1 gene inhibit adipocyte differentiation in ALDH1A1-/- preadipocyte. |
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