| Buffalo is rich in China.However,due to its low intramuscular fat content,the tenderness and flavor of beef is far less than that of beef from cattle.Therefore,it is important to improve the meat performance,to discover vital regulator of fat deposition,and to improve the meat quality in buffalo.Recently,studies show that lone none coding RNA(lnc RNA)plays important role in adipogenesis.Therefore,subcutaneous adipose tissues with vaviable monthes were used for RNA-seq to reveal differentially expressed(DE)lnc RNAs and m RNAs in buffalo.Co-expression analysis and functional enrichment for DE lnc RNAs and m RNAs were performed to obtain putative lnc RNAs involeved in fat deposition.Further,q RT-PCR validation,RACE analysis,cell localization,and overexpression experiments were used to estimate the effect of putative lnc RNAs in adipogenesis in buffalo.The main results are as follows:(1)A total of 133,216,720 to 224,667,746 raw reads data and 130,254,066 to217,117,252 net reads were obtained.More than 76% of the reads in each sample can be mapped to cattle genome.There 21,693 m RNAs were identified.Among them,2,506 m RNAs were DE in 6-month-old and 30-month-old adipose tissue,including 1,292 m RNAs up-regulated and 1,214 m RNAs down-regulated in 30-month-old adipose tissue compared to 6-month-old adipose tissue.(2)There 62,019 lnc RNAs were identified.Among them,proportion of intergenic lnc RNA was the highest(79%).DE analysis yielded 479 lnc RNAs,including 298up-regulated and 181 down-regulated in 30-month-old compared with 6-month-old adipose tissue.(3)DE m RNAs were uesed for GO and KEGG analysis to yield 34 metabolic processes and 8 signaling pathways related to fat deposition(P < 0.05),containing 139 and 281 genes(m RNA),respectively.In total,380 genes were obtained.These 380 DE m RNAs and 479 DE lnc RNA were used for co-expression analysis,and 1958 pairs of fat deposition-related lnc RNA-m RNA(absolute value r > 0.90)were obtained.(4)Two lnc RNAs significantly associated with fat deposition were identified by q RT-PCR and 50 Xinyang buffalo adipose tissue samples,TCONS＿00539092(absolute value r = 0.81)and TCONS＿00539210(absolute value r = 0.78).The full-length of TCONS＿00539092 was 2493 bp and the TCONS＿00539210 was 556 bp.The results of RNA nucleoplasm analysis indicated that TCONS＿00539092 was mainly in the nucleus,and TCONS＿00539210 was detected in both of the nucleus and cytoplasm.Results of expression profiling showed that the expression levels of these two lnc RNAs were significantly higher in adipose tissue than that in other tissues.Meanwhile,they showed increased expression with the induction of differentiation in bovine adipocytes.(5)Overexpression of TCONS＿00539092 and TCONS＿00539210 promoted lipid accumulation in bovine primary adipocytes(P < 0.05),and promoted the expression of adipogenic marker genes C/EBPα,PPARG and other metabolic related genes THRSP and LPL(P < 0.05).In summary,this study used RNA transcriptome sequencing analysis to obtain a large number of m RNA and lnc RNA DE in buffalo adipose tissue at 6 months and 30 months old.By a series of screening,identification,and functional analysis,TCONS＿00539092 and TCONS＿00539210 promotes lipid accumulation in bovine primary adipocytes,but the specific regulatory mechanisms remain to be further studied.This study provides an important basis for further research on the mechanism of lnc RNA regulation of buffalo fat deposition,and provides important information for the improvement of buffalo quality. |
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