Comparative Transcriptomics Of Long Non-coding RNA Related To Tail Fat Deposition Of Sheep

Lanzhou Fat-Tail sheep(LFTS)is a famous local breed in China.It is now in an endangered and its excellent genetic resources are in urgent need of protection.Compared with the traditional local breeds,Small Tail Han sheep(STHS)and Tibetan sheep(TS),the most significant difference is the characteristics of fat tail of LFTS.The fat is an important tissue for storing energy.The tail fat of sheep is the tissue that specifically stores fat.The tail fat of sheep can not only help them resist the harsh environment,but also are necessary energy resource.In recent years,more and more studies have been done on the tail fat development of sheep,mainly from the aspects of genome-wide association analysis,mRNA sequencing,and miRNA sequencing.However,there are few studies on lncRNA.Therefore,in order to explore the regulatory factors associated with fat deposition and development in the tail of sheep,nine individuals from three varieties of LFTS(n=3),STHS(n=3)and TS(n=3)were strand-specific RNA sequencing in this study.Differentially expressed mRNA and lncRNA among the different breed were screened,and through enrichment analysis and network construction,some mRNA and lncRNA that be related to tail fat development were obtained,and its were experimental verification.These studies will enhance researcher understanding of the complex molecular mechanisms of tail fat deposition and provide evidence for the further study of related genes and functional mechanisms.1.Screening and identification of differentially expressed mRNA in tail fat of sheep1.1 Screening,functional enrichment and identification of differentially expressed mRNA in tail fat of sheepThis study performed strand-specific RNA sequencing of nine individuals from three different sheep breeds: LFTS(n=3),STHS(n=3)and TS(n=3).A total of 24,940 known genes were detected in the three breeds,of which 23,596 were detected in the LFTS,22,228 were detected in the STHS,and 22,795 were detected in TS.By comparing each pair,407 differentially expressed genes(DEG)were identified from the three comparison groups.Among them,there are 10 DEG between LFTS and STHS(7 up-regulated genes and 3down-regulated genes),and 390 DEG between LFTS and TS(215 up-regulated genes and 175down-regulated genes).There are 40 DEG(39 up-regulated genes and 41 down-regulated genes)between STHS and TS.Finally,it was found that some DEG were enriched in functions or pathways related to fat deposition(such as fatty acid elongation or fatty acid metabolism)by enriching the function of the DEG of tail fat and participating signal pathways.Fourteen differentially expressed genes were selected from the analysis results of different comparison groups.Through q RT-PCR experiments,it was found that there was a significant difference in DPT and RASD1 between tail fat of LFTS and STHS;In tail fat of LFTS and TS,PDK4 and PLIN2 were significantly different;In the comparison of tail fat of STHS and TS,the expression of SLC22A4 was significantly different.Other genes(FMO2,PENK,MID1IP1,PRKAR2 B,ELOVL3,TCAP,LTF,ADGRG3 and LEPR)were not significantly different,but the expression trends were consistent with the RNA-Seq results,indicating that these genes were related with tail fat deposition in sheep.1.2 Identification of genetic variation related to tail fat of sheep based on mRNA sequencing dataBased on the mRNA sequencing data of tail fat of sheep,the genetic variations of the three breeds were analyzed.A total of 7,122,920 SNPs were detected,of which 14.5% were novel sites.Among the total SNPs,105,024 were located in open reading frames,35.6% of which corresponded to nonsynonymous or stop-gain/stop-loss codons and thus may be functional.Some traits related to tail fat were retrieved from the Animal Quantitative Trait Loci Database(Animal QTLdb)database.It was found that 8.5% of all SNPs and INDELs were located in the QTL region associated with possible functions related to tail fat deposition.Further studies revealed 26,613 specific SNPs in LFTS,and compared with previous reports,44 SNPs were concordant with the previous studies(such as CREB1)performed in other sheep breeds.It shows that some genes may be related to tail fat deposition in sheep.2.Screening of differentially expressed lncRNAs of tail fat of sheep,prediction of target genes,and functional enrichment analysis and identificationBy strand-specific RNA sequencing of three different sheep breeds,9,082 lncRNAs were found,of which 8,929 were detected in the tail fat of LFTS,8,776 were detected in tail fat of STHS,8,901 were detected in tail fat of TS.By analysis,68 differentially expressed lncRNAs were screened from the three comparison pairs.Among them,37 differentially expressed lncRNAs(16 up-regulated lncRNAs and 21 down-regulated lncRNAs)were found between LFTS and STHS.59 differentially expressed lncRNAs(31 up-regulated lncRNAs and 28down-regulated lncRNAs)were found between LFTS and TS.There are 16 differentially expressed lncRNAs(8 up-regulated lncRNAs and 8 down-regulated lncRNAs)between STHS and TS.Then,the target genes of lncRNAs were predicted from both cis and trans,and the predicted target genes were enriched.It was found that some of target genes of the differentially expressed lncRNAs are enriched in some functions or pathways related to fat development.Six differentially expressed lncRNAs were selected from the analysis results of different comparison groups.Through q RT-PCR experiments,it was found that the expression of ENSOART00000027984 was significantly different between tail fat of LFTS and STHS;Between the tail fat of LFTS and TS,although the results were not significant,the expression trends were consistent with the RNA-Seq results;in the comparison of tail fat of STHS and TS,the expression of ENSOART00000028008 was significantly different.This shows that these lncRNAs can affect the development of tail fat in sheep.3.Network construction based on differentially expressed mRNA and lncRNA in tail fat of sheepUsing the screened differential expression mRNA and lncRNA of tail fat of sheep to co-expression analysis,493 pairs of significant co-expression pairs were finally obtained,and most of which were positively correlated(475 pairs)and a few of which were negatively correlated(18 pairs).Finally,using the screened mRNA-lncRNA pairs to construct a co-expression network,it was found that some lncRNAs interact with more than 50 mRNA,for example,67 mRNA co-expressed with TCONS＿00372767 、 TCONS＿00171926 and TCONS＿00054953,respectively,and 65 mRNA co-expressed with TCONS＿00373007,indicating that these lncRNAs belong to the core lncRNAs and have important regulatory effects on tail fat deposition.