| Circular RNA(circRNA)is a kind of widespread and stable existence in animal,plant and microbial cells.It is related to functions such as variable shearing,translation regulation,mi RNA sponge and formation of various tissues.There are few reports on the specific circRNAs in Ningxiang pigs.However,little is known about what their possible functions are,whether they are conserved,and what the temporal and spatial expression patterns of these circRNAs during tissue development are.The selected 12 male Ningxiang pigs(30d,90 d,150d and 210 d after birth)were subjected to circRNA sequencing and analysis as the research object.Foure groups of pig are half-sibs,and the duplicate samples are full-sibs.RNA-seq,mi RNA-seq,Label Free and Parallel Reaction Monitoring(PRM)were employed to analyze the transcriptome and proteome of pig liver tissue in three critical development periods(lactation,nursery and fattening),and identify the key genes and mi RNAs related to liver development and fatty acid synthesis and metabolism.This study have explored the transcriptional regulation mechanism of Ningxiang pig liver development and fatty acid metabolism.It will provid a theoretical basis for the discovery of pork quality functional genes and molecular breeding applications.(1)Circ RNA-seq analysis of three different tissues of Ningxiang pigs at four different time points showed that a total of 516.27 GB of clean data was obtained,and the clean data of each sample was more than 11.7 GB,and the percentage of Q30 bases was more than94.36%.A total of 61,683 circRNAs were identified by the CIRI and find＿cir soft-wares.(2)In the circRNA-Seq analysis of longissimus dorsi,349,362,and 452 differentially expressed circRNAs(DECs)(P-value <0.05 & |log2FC| >1)were identified in the N30 d vs N90 d,N30d vs N150 d,and N30 d vs N210 d groups at the closed time point group,55 of them DECs are co-expressed;In addition,417 and 206 cases were identified in the N90 d vs N150 d and N150 d vs N210 d groups at consecutive time points.Short Time-Series Expression Miner(STEM)analysis showed that these circRNAs were significantly clustered in five expression patterns(p<0.05).(3)In the circRNA-Seq analysis of subcutaneous fat,475,936 and 937 DECs were identified in the N30 d vs N90 d,N30 d vs N150 d,and N30 d vs N210 d groups at the closed time point group,256 of them DECs are co-expressed.In addition,580 and 188 DECs were identified in the N90 d vs N150 d and N150 d vs N210 d groups at consecutive time points.STEM analysis showed that these circRNAs were significantly clustered in six expression patterns;(4)In the circRNA-Seq analysis of liver,1015,986 and 937 DECs were identified in the N30 d vs N90 d,N30 d vs N150 d,and N30 d vs N210 d groups at the closed time point group,559 of them DECs are co-expressed.Only 234 and 190 DECs were identified in the N90 d vs N150 d and N150 d vs N210 d groups at consecutive time points.STEM analysis showed that these circRNAs were significantly clustered in seven expression patterns;(5)Combined with STEM and ce RNA analysis,2384(fat),2672(muscle)and 9187(liver)circRNAs-mi RNAs-m RNAs network pairs related to developmental time changes were identified.Multiple ce RNA network pairs related to cell cycle,cell differentiation,and fatty acid biosynthesis metabolism markers were screened out;(6)Based on the physiological and nutritional characteristics of Ningxiang pig,a total of 5624 peptides and 1840 proteins expressed from medium to high abundance were identified by Label-free analysis of liver tissue in three critical periods(30 d,90 d and 210 d after birth).There were 192 differential expressed proteins(DEPs)in N30 d vs N90 d,of which 88 were up-regulated and 104 were downregulated.There were 268 DEPs in N30 d vs N210 d,of which 72 were upregulated and 196 were down-regulated.N90 d vs N210 d had293 DEPs,of which 75 were upregulated DEPs and 215 were down-regulated DEPs(Fold change ≥ 1.2 or ≤ 0.8 and P < 0.05);In addition,29,55 and 132 DEPs were found to be differentially expressed in N30 d vs N90 d,N30 d vs N210 d and N90 d vs N210 d,respectively.17 DEPs were co-expressed in the three groups.Furthermore,the upregulated DEPs in N30 d vs N90 d were significantly enriched in 63 KEGG pathways,including disease immunity,amino acid metabolism and fatty acid degradation.In N90 d vs N210 d group,up-regulated DEPs were only significantly enriched in Protein and absorption.Meanwhile,DEPs downregulated in the N30 d vs N90 d group were significantly enriched only in the HIF-1 signaling pathway,glucagon signaling pathway,ECM-receptor interaction and pentose phosphate pathway,which are related to the control of cell proliferation and differentiation and maintenance of cellular glucose metabolism homeostasis,while the significantly downregulated pathways in the N90 d vs N210 d group were mainly involved in the control of RNA transcription and protein translation.During the development of liver tissue that DEPs are downregulated(eg CD36,HSPA5 and FBP1,etc.)related to fatty acid oxidative deposition were also identified and DEPs are upregulated(eg CD26,AUH,CDH1 and ECHDC1,etc.)related to cell development and lipid metabolism.(7)Combining analysis of the liver proteome with mi RNA and transcriptome,2993 mi RNA-m RNA interaction negative regulatory pairs in the liver were found,involving 54 mi RNAs(of which 31 mi RNAs were newly identified)and 2138 m RNAs.For example,ssc-mi R-4332-UCP3,Chr08＿21275-ACACA,ssc-mi R-885-3p-CCN4,ssc-mi R-4332-ATP2A3,ssc-mi R-874-LDLRAD4,and ssc-mi R-138-LDLRAD3 were associated with lipid anabolism.Protein interaction network analysis showed that UROD might acted as a "hub" protein,which was located at the important nodes of protein network related to liver development and fat deposition metabolism,together with SOD1,LDHA and PON2,respectively.The related mi RNA-m RNA regulatory pair could serve as a potential major network pair in the development of liver tissue.In summary,this study analyzed the differences in circRNA expression at 4 different developmental time points in 3 different tissues of Ningxiang pig through a comprehensive comparison.Combining liver transcriptome and proteomic will lay the foundation for elucidating the regulatory molecular mechanisms of different tissue development and pork quality traits in Ningxiang pig. |
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