Study On Tail Fat And The Role Of OXCT1 In Differentiation Of Adipocyte In Tan Sheep

Tan sheep is a famous meat and fur sheep breed with long and fat tail.It is mainly distributed in the bordering areas of the three provinces(regions)of Shaanxi,Gansu and Ningxia,where is also a desert or semi-desert ecological zone and a farming-pastoral ecotone.The large amount of fat stored in the tail of Tan sheep is a necessary energy reserve for living in the cold winter at the tranditional grazing.However,the breeding of Tan sheep has changed from grazing to house feeding and the production use has shifted from fur to meat at present.The fat tail of Tan sheep has also become disadvantages instead of early advantages.As a leading breed of high-quality mutton sheep,Tan sheep must be an important research topic on how to achieve “slimming”,reduce tail fat,impove carcass quality and feed utilization.In this study,transcription factors(C/EBP?)and key enzyme genes(FAS,LPL,and HSL)that play an important role in fat deposition were selected to comare their expressions in tail fat of Tan sheeps with different dietary nutrition levels and growth stages.Correlationships between the expressions and tail fat phenotypic data were analyzed.Another different expression of the four genes in different part fat and growth stages of Tan sheep was detected to explain the regulation further.To find the genes closely reated to tail fat deposition,this study analyzed the correlations between the tail phenotype of Tan sheep and Hedgehog signaling pathway as well as the ketone decomposition key enzyme gene,OXCT1.On this basis,OXCT1 was selected to research in adipocyte from Tan sheep tail fat and explore its role in adipocyte differentiation further.The main results obtained in this study are as follows:1.In the tail fat of Tan sheep fed with different nutritional diets(84 %,96 %,108 %,and 120 % of the standard diets)at three growth stages(i.e.,each stages last one month and took 22 to 28 kg,29 to 35 kg,and 36 to 40 kg as weight gain targets for stages 1,2,and 3,respectively),the m RNA expression levels of C/EBP? and FAS genes increased first and then decreased with the increase of dietary nutrion,and the highest expression was in 108 % treatment(P<0.05).The m RNA expression level of LPL and HSL decreased with the increase of dietary nutrion(P<0.05).The result of two-way ANOVA showed that dietary nutrition significantly affected the expression of C/EBP?,and the same of dietary nutrition and growth stage to FAS,LPL and HSL m RNA expressions(P<0.05).The interactions of dietary nutrition and growth stage significantly affected the expressions of above all genes.By reanalyzing the tail phenotypic data of Tan sheep(variations of tail length,width and thickness at each stages,as well as the tail weight at the end of each stage)and correlation analyzing the relationship between tail phenotype and gene expression,C/EBP?,FAS and LPL were correlated with the change of tail phenotype and the 108 % of the standard diets was the better choice.2.Considering the excellent feeding effect,the Tan sheep with 108% of the standard diets were selected to be test sheep,and their tail,subcutaneous,and perrenal adipose tissue were collected for further experiment.In general,the m RNA expressions of C/EBP? and FAS in tail fat at each stage were significanty higher than those in subcutaneous and perrenal fat(P<0.05).while LPL and HSL m RNA expressions were significantly highest in subcutaneous and perrenal fat,respectively(P<0.05).According to the result of two-way ANOVA,different part fat had a significantly effect on the expression of C/EBP? and FAS,and the same of growth stages on HSL expression(P<0.05).The interaction of different part fat and growth stage significantly affected the expression of FAS,LPL and HSL(P<0.05).The main effect analysis showed that the expression of C/EBP? in Tan sheep tail fat was significantly higher than that of subcutaneous and perrenal fat(P<0.05);there was no dsignificant difference between subcutaneous and perrenal fat(P > 0.05).It showed that the expression patterns of fat-related genes in different part fat from Tan sheep were different.3.In the tail fat of Tan sheep with different nutrient at three growth stages,the m RNA expressions of IHH,GLI1,SMO,and PTCH1 in Hedgehog signaling pathway decreased significantly with the increase of dietary nutrition(P<0.05);OXCT1 m RNA expression increased first and then decreased with the increase of dietary nutrition,and the highest expression was in 108 % treatment(P<0.05).The result of two-way ANOVA showed that dietary nutrition significantly affected the expression of IHH,SMO,PTCH1 and OXCT1,and the same of growth stage on the expression of IHH and GLI1.The interactions of dietary nutrition and growth stage significantly affected the expression of IHH and PTCH1(P<0.05).The main effect analysis showed that the expression of GLI1 increased significantly with growth stages,and GLI1 and SMO decreased significantly with the increase of dietary nutrition(P<0.01).The m RNA expression of OXCT1 in the four different nutritional diets increased first and then decreased with the increase of dietary nutrition,and the highest expression was in 108 % treatment(P<0.001).According to the correlation analysis between the gene expressions and tail phenotypic data,Hedgehog signaling pathway and OXCT1 had certain correlation with the tail phenotype of Tan sheep.4.The above results show that OXCT1 was one of the genes related to the deposition of tali fat in Tan sheep.Therefore,a series of experiments were carried out around OXCT1.At first,we successfully cloned the CDS of ovine OXCT1 and predicted its protein sequence.The comparison of the predicted protein sequence with cattle,pigs,mice and humans demonstrated a high degree of homology in OXCT1.Secondly,the result of different tissue expression partern showed that OXCT1 was widely expressed in different tissues of Tan sheep,mainly in kidney,small intestine,lung and spleen tissues,followed by tail adipose heart,stomach,liver and cartilage tissues,but not in the longissimus dorsi muscle.Thirdly,the preadipocytes were successfully isolated from the tail adipose of Tan sheep and induced into mature adipocytes with obvious lipid droplets and rings.During the differentiation,the expression of OXCT1 in adipocytes during the 8-days of induction increased from the day 1 to day 6,but decreased markerdly on the day 8.It shows that OXCT1 functions in the mid-period of adipocyte differentiation in vitro from Tan sheep tail.Fourthly,the lentiviral overexpression and interference vectors were successfully constructed and packaged to lentivirus.Lentivirus infected preadipocytes to obtain stable passaged cell lines.The expressions of PPAR?,C/EBP? and LPL increased and HSL decreased in OXCT1 overexpression preadipocytes from Tan sheep;lipid accumulation and differentiation of adipocytes were inhibited.The expressions of genes,lipid accumulation and adipocytes differentiation in OXCT1 interference preadipocytes from Tan sheep showed converse results.It indicated that OXCT1 was negatively related to tail fat deposition in Tan sheep.