No abstract

To provide new ideas for improving meat quality and generating new breeds of cattle, the important candidate genes affecting fat deposition in two kinds of cattle were identified. Eighteen months Shandong black cattle (n = 3) and Luxi cattle (n = 3) were randomly assigned into two environmental. The longissimus dorsi muscles of Shandong Black Cattle and Luxi Cattle were collected and analyzed by fatty acid determination, high-throughput sequencing transcriptomics, qRT-PCR expression profile and western blot. The ratio of unsaturated fatty acids to saturated fatty acids was 1.37:1 and 1.24:1 in the muscle tissues of Shandong black cattle and Luxi cattle, respectively. The results of RNA-Seq analysis revealed 1320 DEGs between the longissimus dorsi of Shandong black cattle and Luxi cattle. A total of 867 genes were upregulated, and the other 453 genes were downregulated. With GO enrichment analysis, it was found that the identified DEGs were significantly enriched in regulation of the Wnt signaling pathway, negative regulation of the Wnt signaling pathway, cAMP metabolic process, fat cell differentiation and among other functions. We found that regulation of lipolysis in adipocytes was the significant enrichment pathway of upregulated genes and downregulated genes, PPAR signaling pathway and AMPK signaling pathway are highly representative pathways of lipid metabolism in Shandong black cattle. Network analysis showed that PPARGC1A, ADCY4, ANKRD6, COL1A1, FABP4, ADIPOQ, PLIN1, PLIN2, and LIPE genes were correlated with key loci genes in multiple metabolic pathways. Meanwhile we found that FABP4 and ADIPOQ had 7 common regulatory factors in different genes, which were PLIN1, PLIN2, PPARGC1A, RXRA, PCK1, LEPR, LEP. These genes were involved in regulation of lipolysis in adipocytes, adipocytokine signaling pathway, PPAR signaling pathway. FABP4 and ADIPOQ were selected as important candidate marker genes for fat deposition based on the results.

Shandong black cattle is a new breed of cattle that is developed by applying modern biotechnology, such as somatic cloning, and conventional breeding methods to Luxi cattle. It is very important to study the function and regulatory mechanism of circRNAs in muscle differentiation among different breeds to improve meat quality and meat production performance and to provide new ideas for beef cattle meat quality improvements and new breed development. Therefore, the goal of this study was to sequence and identify circRNAs in muscle tissues of different breeds of cattle. We used RNA-seq to identify circRNAs in the muscles of two breeds of cattle (Shandong black and Luxi). We identified 14,640 circRNAs and found 655 differentially expressed circRNAs. We also analyzed the classification and characteristics of circRNAs in muscle tissue. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used on the parental genes of circRNAs. They were mainly involved in a variety of biological processes, such as muscle fiber development, smooth muscle cell proliferation, bone system morphogenesis, tight junctions and the MAPK, AMPK, and mTOR signaling pathways. In addition, we used miRanda to predict the interactions between 14 circRNAs and 11 miRNAs. Based on the above assays, we identified circRNAs (circ0001048, circ0001103, circ0001159, circ0003719, circ0003424, circ0003721, circ0003720, circ0001519, circ0001530, circ0005011, circ0014518, circ0000181, circ0000190, circ0010558) that may play important roles in the regulation of muscle growth and development. Using real-time quantitative PCR, 14 circRNAs were randomly selected to verify the real circRNAs. Luciferase reporter gene system was used to verify the binding site of miR-1 in circ0014518. Our results provide more information about circRNAs regulating muscle development in different breeds of cattle and lay a solid foundation for future experiments.

Fat metabolism is closely related to the economic characteristics of beef cattle. Therefore, regulating fat deposition and increasing intramuscular fat deposition are among the main goals of breeders. In this study, we aim to explore the regulatory role of CB1 gene on PPARγ2/PLIN1/HSL pathway in fat metabolism, and to further explore the differential expression of regulatory factors of this pathway in Shandong black cattle and Luxi cattle. In this study, CB1 overexpression stimulated lipid synthesis in adipocytes to some extent by increasing the levels of FASN and ACSL1. CB1 inhibitors reduce the lipid content in adipocytes and reduce the expression of GLUT1 and Insig1. In addition, overexpression of CB1 decreased the expression of PPARγ2 and led to an increase in PLIN1 expression and a decrease in HSL expression in adipocytes. We also found that the CB1/PPARγ2/PLIN1/HSL was differentially expressed in the different breeds of cattle and was involved in the regulation of fat metabolism, which affected the fatty acid content in the longissimus dorsi muscle of the two breeds. In short, CB1 participates in lipid metabolism by regulating HSL in the PPARγ2 and PLIN1 pathways, and improves lipid formation in adipocytes. In conclusion, CB1/PPARγ2/PLIN1/HSL pathway may be involved in the regulation of lipid metabolism.

There is an increasing understanding of the possible regulatory role of long non-coding RNAs (LncRNA). Studies on livestock have mainly focused on the regulation of cell differentiation, fat synthesis, and embryonic development. However, there has been little study of skeletal muscle of domestic animals and the potential role of lncRNA. In this study, the transcriptome numbers of longissimus muscle of different beef cattle (Shandong black catle and Luxi catlle) were used to construct muscle related lncRNAs-miRNA-mRNA interaction network through bioinformatics analysis. This is helpful to clarify the molecular mechanism of bovine muscle development, and can be used to promote animal husbandry and improve animal husbandry production. According to the screening criteria of |FC|≧2 and q < 0.05, a total of 1,415 transcripts (of which 480 were LncRNAs) were differentially expressed (q < 0.05) in the different breeds. Further, we found that the most differentially expressed LncRNAs were found on chromosome 9, in which the differentially expressed LncRNAs targeted 1,164 protein coding genes (MYORG, Wnt4, PAK1, ADCY7,etc) (upstream and downstream<50 Kb). In addition, Pearson’s correlation coefficients of co-expression levels indicated a potential trans regulatory relationship between the differentially expressed LncRNAs and 43844 mRNAs (r > 0.9). The identified co-expressed mRNAs (MYORG, Dll1, EFNB2, SOX6, MYOCD, and MYLK3) are related to the formation of muscle structure, and enriched in muscle system process, strained muscle cell differentiation, muscle cell development, striated muscle tissue development, calcium signaling, and AMPK signaling. Additionally, we also found that some LncRNAs (LOC112444238, LOC101903367, LOC104975788, LOC112441863, LOC112449549, and LOC101907194) may interact with miRNAs related to cattle muscle growth and development. Based on this, we constructed a LncRNAs-miRNA-mRNA interaction network as the putative basis for biological regulation in cattle skeletal muscle. Interestingly, a candidate differential LncRNA (LOC104975788) and a protein-coding gene (Pax7) contain miR-133a binding sites and binding was confirmed by luciferase reporter assay. LOC104975788 may combined miR-133a competitively with Pax7, thus relieving the inhibitory effect of miR-133a on Pax7 to regulate skeletal muscle development. These results will provide the theoretical basis for further study of LncRNA regulation and activity in different cattle breeds.