Identification Of MiRNAs And LncRNAs Of Tail Fat Differences In The Second Generation Of Wild Ari Crosses

In this study,the small tail type of the second generation of cross between Ari and Bashibai sheep(F2sheep)was selected as the research object,and Bashibai sheep was used as the control.The tail tissues of the two populations were collected for whole transcriptome sequencing,and we used q RT-PCR to verify the results of transcriptome sequencing.Differential expression of miRNAs and lncRNAs and their involved signaling pathways were screened by bioassay.Then four miRNAs and four lncRNAs related to fat deposition and their target genes were selected.The expression characteristics of miRNAs,lncRNAs and their target genes in 12 tissues including tail fat,subcutaneous fat,longissimus dorsi,brachial triceps,quadriceps femoris,heart,liver,spleen,lung,kidney,perirenal fat and intestinal fat of Bashibai sheep and F2 sheep were quantitatively analyzed by q RT-PCR.Furthermore,the function prediction analysis of fat deposit-related miRNAs,lncRNAs and their target genes was conducted to provide a basis for revealing the regulatory mechanism of tail fat metabolism in sheep.The main results are as follows:1.Identification and analysis of miRNAs and lncRNAs obtained byRNA-seq sequencing:Six pieces of tail fat were collected from fat-tailed bashibai sheep and six pieces of tail fat from smalltailed F2 sheep,andRNA-seq was performed.After filtration,a total of 698,963,120 clean reads were obtained,accounting for 97.18% of high-quality sequence data.The average ratio of comparison efficiency between original sequence data and reference genome was 90.41%.Through differential expression analysis,125 differentially expressed miRNAs and 728 lncRNAs were selected,among which 49 miRNAs and 270 lncRNAs were down-regulated,and 76 miRNAs and 458 lncRNAs were up-regulated.GO analysis showed that it was mainly involved in protein transport,transcriptional regulation,muscle contraction,mRNA regulation and information transmission.KEGG pathway analysis revealed that it may be involved in the regulation of immunity and energy metabolism.Four differentially expressed miRNAs and lncRNAs were screened out for q RT-PCR,and the results were consistent withRNA-seq results,proving the authenticity of transcriptomic data.2.Expression differences and functional prediction of tail fat deposit-related miRNAs:Four differentially expressed miRNAs(miR-24-3p,miR-29 a,miR-30 b and miR-193b-3p)and target genes(SEC31B,GPAM,TADA2 A and DGAT2)were screened by bioassay,and relative quantitative analysis was performed in tissues of two different populations.The results showed that the expression pattern of target genes in each tissue of Bashibai and F2 sheep was opposite to that of miRNAs,suggesting that miRNAs regulated tail fat deposition by inhibiting the target genes.After combined analysis of differential miRNAs and their target genes,there were significant and extremely significant differences in tail fat between miR-24-3p and SEC31 B,miR-29 a and GPAM,miR-30 b and TADA2 A,miR-193b-3p and DGAT2,mainly in Bashibai sheep and F2 sheep.SEC31 B participates in PPARγ signaling pathway to regulate adipocyte differentiation,and miR-24-3p is highly expressed in F2 goat tail fat(P<0.01),while SEC31 B was significantly lower in F2(P<0.01),it was speculated that miR-24-3p directly positively regulates fat deposition in sheep through SEC31B;GPAM is a key gene of phospholipid biosynthesis and triglyceride synthesis.The expression of miR-29 a in F2 sheep liver was significantly higher than that in Basbai sheep(P<0.01),while GPAM was significantly lower in F2 sheep liver(P<0.01),it was speculated that miR-29 a indirectly and positively regulates fat deposition in sheep through the involvement of GPAM in lipid synthesis;TADA2A mainly encodes lysine acetyltransferase,which is a key factor of energy metabolism.miR-30 b was significantly overexpressed in longissimus dorsi muscle and triceps brachium muscle of F2 sheep(P<0.05)and was significantly lower in quadriceps femoris(P<0.05),and the expression pattern of TADA2 A in intermuscular fat was completely opposite to that of miR-30 b,suggesting that miR-30 b indirectly positively regulates fat deposition in sheep through TADA2A’s involvement in intermuscular fat energy metabolism.DGAT is a rate-limiting enzyme involved in the synthesis of triglycerides,and is involved in the absorption of fat in the intestinal tract.miR-193b-3p is extremely low in the peritroid lipid of F2 sheep(P<0.01),DGAT2 was significantly higher in F2,and it was speculated that miR-193b-3p indirectly and negatively regulated sheep tail fat deposition by participating in intestinal lipid synthesis through DGAT2.3.Expression differences and functional prediction of tail fat deposit-related lncRNAs:Four differentially expressed lncRNAs(MSTRG.38164,MSTRG.36912,MSTRG.8169 and MSTRG.31389)and target genes(GPAM,NDUFC2,WDTC1 and PIK3R1)were screened by bioassay.The results showed that the expression trend of target genes in tissues of Bashibai and F2 sheep was the same as that of lncRNAs,indicating that lncRNAs affected tail fat deposition by positively regulating the expression of target genes.After combined analysis of differential lncRNAs and their target genes(MSTRG.36912 and NDUFC2,MSTRG.8169 and WDTC1,MSTRG.38164 and GPAM,MSTRG.31389 and PIK3R1),the expression patterns of all of them in different tissues of Bashibai and F2 sheep were basically similar,and there were significant and extremely significant differences in tail fat.NDUFC2 was closely related to adipocyte differentiation.Compared with Bashibai sheep,MSTRG.36912 and its target gene NDUFC2 were significantly lower in F2 sheep tail fat(P<0.01),MSTRG.36912 May directly and positively regulate fat deposition in sheep tail through NDUFC2;WDTC1 was a lipid accumulation suppressor gene,MSTRG.8169 and its target gene WDTC1 were significantly overexpressed in the tail fat of F2 sheep(P<0.05),it was speculated that MSTRG.8169 directly and negatively regulated sheep tail fat deposition through WDTC1.GPAM is a key gene in phospholipid biosynthesis and triglyceride synthesis.MSTRG.38164 and its target gene GPAM were significantly lower in the liver of F2 sheep(P<0.01),it is speculated that MSTRG.38164 indirectly and positively regulate fat deposition in sheep by affecting lipid synthesis through GPAM.PIK3R1 is closely related to insulin action.MSTRG.31389 and its target gene PIK3R1 were significantly overexpressed in brachial triceps,quadriceps femoris and liver of F2 sheep(P<0.05),suggesting that MSTRG.31389 indirectly and negatively regulate sheep tail fat deposition through PIK3R1 affecting intermuscular and liver metabolism.Conclusion: miRNAs(miR-24-3p,miR-29 a,miR-30 b,and miR-193B-3p)and lncRNAs(MSTRG.38164,MSTRG.36912,MSTRG.8169,and MSTRG.31389)exert lipid regulation by regulating downstream target genes Among them,miRNAs negatively regulate target genes and lncRNAs positively regulate target genes,which directly or indirectly affect sheep tail fat deposition,thus causing the difference in sheep tail type.