Whole Genome Profiling Of MicroRNAs Expressed In Tail Fat And Their Associations With Traits In Sheep

Content of body fat in domestic animals and poultry determines the meat quality. Fat metabolism is a complex biological process regulated by many factors. MicroRNAs (miRNAs) are small non-coding RNA molecules (sncRNA) with the length of ～22 nt. These miRNAs involve in biological processes, e.g. cell differentiation, growth and development and performance expression, etc. by regulating their target mRNAs via complete or incomplete base matching at the post-transcriptional level. Understanding the regulating functions of fat metabolism-related miRNAs can, to a certain extent, reveal the molecular regulation mechanism of fat metabolism. However, no study was found about the regulation function of miRNAs expressed in tail fat of sheep.In order to better understand the role of miRNAs in fat metabolism, two small RNA libraries were constructed and high-throughput sequencing was used to identify the miRNAs expressed in the tail fat of two sheep breeds with distinct tail type (Guangling fat-tailed sheep and small tailed Han) and to screen the differentially expressed miRNAs. Bioinformatics methods were used to predict their target genes, which were subsequently subject to Gene Ontology and KEGG analysis. Six miRNAs (miR-10b, miR-29a, miR-30c, miR-155, miR-192 and miR-206) based on the sequencing results were chosen to verify the sequencing results by detecting their expressions in the animals at 10 months of age using quantitative real time PCR technique. The same technique was also used to detect the expressions of the six miRNAs in tail fat of the two breeds in six age groups (2,4,6,8,10,12 month of age). Effects of breed, sex, month of age and two-way interactions were estimated by fitting general linear models. Pearson’s correlation coefficients were computed to study the associations between miRNA expressions and tail and slaughter traits. The main results were as follows:1. Total of 113 and 131 conservative miRNAs with 22890 and 22907 target genes in the tail fat of Guangling Fat-tailed and Small Tailed Han sheep were obtained, respectively. The number of miRNAs expressed differentially in the two breeds was 93,58 of which expressed significantly. By KEGG analysis on the regulation network of the target genes,309 pathways, including Wnt, MAPK, were found in the two libraries. By screening the unknown miRNAs using miReap,208 and 215 novel miRNAs with 22927and 22929 target genes in the two libraries were obtained, respectively. Aligned these novel miRNAs with the conservative ones of other species in miRBase 21.0, most of the novel ovine miRNAs can be found in the database, and there were 27 novel miRNAs for all species. The number of novel miRNAs expressed differentially in the two breeds is 175,105 of which expressed significantly.2. The result of quantitative real-time PCR showed that the six miRNAs differentially expressed significantly in the two breeds. The trends of miRNA expression were consistent with the sequencing data and implied that these miRNAs involved in fat metabolism in tail fat of sheep.3. Analysis of variance based on the general linear models fitted showed that breed had a significant effect on the expression of the six miRNAs (P<0.01 or P<0.001). Expressions of miR-155 and miR-192 in Small Tailed Han were higher than that in Guangling Fat-tailed, other four miRNAs expressed opposite results. Sex only had a significant effect on miR-206 expression (P<0.05). Breed by sex interaction influenced miR-30c and miR-206 expressions significantly (P<0.01). Significant interaction between breed and month of age was also found for the expression of miR-10b, miR-30c and miR-155 (P<0.01 or P<0.001). Association study indicated that the expression of miR-10b, miR-29a and miR-192 had a positive correlation with all the traits, and the correlations with body weight, carcass weight, absolute and relative tail fat weights were significant (P<0.05 or P<0.01). Significant correlations between miR-29a expression and tail length and dressing percentage were also obtained (P<0.05). The expression of miR-30c also showed a positive correlation with all the traits, but only significant with tail width (P<0.05). Negative correlations with all the traits were found for the expression of miR-155 and miR-206. Particularly, the expression of miR-206 correlated significantly with all traits except dressing percentage (P<0.05 or P<0.01). The associations between miRNA and trait expressions reflected corresponding roles of the miRNAs in fat metabolism.The results of expression profiling of miRNAs in tail fat of Guangling Fat-tailed and Small Tailed Han sheep and association study in this dissertation provide scientific evidences for clarifying the regulation mechanisms of miRNAs involved in fat metabolism, and lay the foundation for further studying the function of target genes in fat metabolism regulated by miRNAs at post-transcriptional level.