| Ruminants have a unique digestive system compared with monogastric animals.Specifically,the gastrointestinal tract of ruminants harbors a large number of microflora that can transform and utilize dietary crude fiber.Traditional studies focus on the complex forestomach of ruminants,while ignoring the study of small intestine and large intestine function.The intestines of ruminants perform unique physiological functions,indicating that molecular regulation in the intestines is far more complicated than previously thought.In recent years,the functions of miRNAs in the intestines have been extensively explored,with many novel miRNAs being identified.MiRNAs have been found to participate widely in the maintenance of intestinal homeostasis and the regulation of metabolic homeostasis.Moreover,miRNAs play an important regulatory role in intestinal mucosal immunity,stress resistance,the development and maintenance of intestinal morphological structure,as well as the regulation of various intestinal functions.It has been reported that miRNAs differentially expressed in differenrt intestinal segements.However,there is a lack of research regarding the miRNAs expression profile in the duodenum,cecum,and colon of sheep.In this study,three individuals were selected from purebred Small-tail han sheep and Dorper sheep under the same feeding conditions.The duodenum,cecum and colon small RNA library were constructed.The six libraries were sequenced using Illumina/Solexa high-throughput sequencing,and the data were analyzed by quality,length distribution statistics,genome localization,sRNAs classification,miRNA identification and family analysis.Differentially expressed miRNAs were screened out between different intestinal tissues,and between different breeds in the same intestine.Gene expression prediction,gene function annotation and KEGG pathway analysis were performed using bioinformatics method,and the differential regulation network between differential miRNA and predicted target genes was constructed.We used qRT-PCR to quantify the differentially expressed miRNAs.The results showed that the high-throughput sequencing results were reliable.The main results of this study are as follows:(1)27,498,891,26,490,229 and 25,351,117 clean reads were obtained by high-throughput sequencing in the duodenum,cecum and colon of Small tail Han sheep,which accounted for 96.51%,95.16% and 97.29% of the total reads respectively.There were22,922,141,25,359,773 and 24,890,242 clean reads in the duodenum,cecum and colon,respectively,accounting for 96.10%,97.81% and 96.72% of the total reading,accordingly.(2)sRNA length distribution statistical analysis found that: sRNA sequence in 6 libraries is mainly distributed between 19-24 nt,the highest proportion is 22 nt sequence,in the Small tail Han sheep,duodenum,cecum,colon accounting for 44.04%,41.48%,33.96%,,respectively,while in Dorper accounting for 34.42%,37.22% and 33.48%.which is followed by 23 nt and 21 nt.(3)Genomic mapping analysis showed that there were 16,733,785 、 17,005,544 、16,567,397 clean reads in duodenum,cecum and colon of Small tail Han sheep matched to the sheep genome,,account for 61%、64%、65% of the total clean reads.There were 13,658,376、14,171,821、16,696,530 clean reads in duodenum,cecum and colon of Dorper sheep matched to the sheep genome,account for 59.6%、55.9%、67.1% of the total clean reads.The most abundent chromosomes of sRNAs are chr5,chr11,chr2.(4)In Small tail Han sheep,128 known miRNAs were identified,526 novel miRNAs were predicted,and 202 differentially expressed miRNAs were found between the different tissues.4,422 candidate target genes were predicted,DAVID analysis showed that 1,070 target genes of the differentially expressed miRNA between duodenum and cecum were enriched into 84 GO entries,30 of which were significantly enriched;581 target genes of the differentially expressed miRNA between duodenum and colon were enriched into 81 GO entries,42 of which were significantly enriched;264 target genes of the differentially expressed miRNA in cecum and colon were enriched into 71 GO entries,38 of which were significantly enriched.KEGG pathway analysis showed that a total of 529 target genes were found to participate in 37 KEGG pathways,21 of which were significantly enriched.The most abundant pathway was metabolic pathway,with a total of 270 target genes(51.3%)annotated into the pathway.A combined regulatory network containing 275 nodes and 781 interactions was constructed based on 206 metabolism-related target genes and 69 differentially expressed miRNAs.The module analysis reveals that the network contains 10 modules,and only one of the modules with scores of more than 9,The module contains a total of 26 nodes,70 interaction,PLC family may play an important role in the module.(5)In Dorper sheep,127 known mi RNAs were identified,469 novel miRNAs were predicted,and 187 differentially expressed mi RNAs were found between 3 intestinal tissues.4,675 candidate target genes were predicted,DAVID analysis showed that 1,065 target genes of the differentially expressed miRNA in duodenum and cecum were enriched into 61 GO entries,of which 27 entries were significantly enriched;506 target genes of the differentiallyexpressed miRNA in duodenum and colon were enriched into 75 GO entries,of which 22 entries were significantly enriched;795 target genes of the differentially expressed miRNA in cecum and colon were enriched into 77 GO entries,of which 36 entries were significantly enriched.KEGG pathway analysis showed that a total of 711 target genes were found to participate in 49 KEGG pathways,of which 28 were significantly enriched.The most abundant pathway was metabolic pathway,with a total of 282 target genes(39.6%)annotated.A combined regulatory network containing 354 nodes and 1,332 interactions was constructed,oar-miR-133 showed the highest interaction value.The module analysis reveals that the network contains 11 modules,and only one of the modules with scores of more than 9,The module contains a total of 27 nodes,88 interaction,11 of 14 target genes were involed in carbon metabolism,7 of which enriched in Glycolysis/gluconeogenesis pathway.(6)205 differentially expressed miRNAs were identified in duodenum,cecum and colon between two sheep breeds.1100 candidate genes with non-redundant and high confidence were predicted.Using DAVID analysis,A total of 728 target genes were enriched into 72 GO entries,of which 566 target genes were significantly enriched to 32 GO entries;in the cecum,69 miRNAs were differentially expressed.A total of 550 target genes were enriched into 27 GO entries,all of which were significantly enriched.In the colon,1100 target genes were predicted from 81 different miRNAs,of which 308 target genes Enriched to 41 GO entries,and 163 target genes were significantly enriched to 23 GO entries.KEGG pathway enrichment analysis of target genes predicted between two different breeds revealed that 427 target genes were annotated into 29 KEGG pathways in the duodenum,14 of which were significantly enriched;In the cecum,a total of 82 target genes were annotated into 7 KEGG pathways,3 of which were significantly enriched;in the colon,a total of 77 target genes were annotated to 14 KEGG pathway,7 of which was significantly enriched.The differences of miRNA and their target genes between different tissues and breeds were analyzed using regulation networks.(7)14 differentially expressed miRNA were chosen to validate the results using qRT-PCR.the results showed that the expression trends of 12 miRNAs were consistent with the sequencing results.Only two miRNAs(miR-200 b and novel-mir-214)showed different trends between the qPCR and sequencing data,all the results suggested that our sequencing was much more reliable. |
PDF Full Text Request