| China Horse Industry is transforming from the traditional horse industry to themodern form. Breeding of new horse lines (breeds) adapt to the natural environmentalconditions of China become an urgent demand. It is necessary to research immune relatedgenes of Mongolian Horse, the excellent local species. There are several reasons to supportthe view above: Firstly, it indicates why Mongolian Horse can strongly resist diseases, andprovides the theoretical proofs to revealing the genetic characteristics of disease resistance;Secondly, the research plays a positive role for protecting, utilizing and breedinginnovation of germplasm; also improves the techniques of the crossbreeding and new lines’cultivation. Mongolian Horse and Thoroughbred have been chosen to be the experientgroup and control group resepectively. First of all, to figure out whether there wassignificant difference between the two species regarding to disease resistance ability, thefollowing blood analysis including biochemical, immune and antioxidant indices at theirnormal biophysical state had been done. Secondly, the study on polymorphism of bothTLR2and TLR4were used to analyze the differences of genotype between the two horsespecies. Thirdly, a differential expression assay of TLRs in different tissues of the twospecies by real-time reverse transcription quantitative PCR (RT-qPCR) had been taken.Meanwhile, stimulating horse monocytes by lipopolysaccharide (LPS), we got the relativeexpression data of immune related genes by the differential expression assay; furtherly, theinfluence of disease resistance genes had been analyzed on the resistance between differentspecies. Finally, expression profile analysis of spleen tissues of Mongolian Horse andThoroughbred by high-throughput sequencing was used to searche the differential genes.This thesis tried to provide the theoretical proofs to revealing the disease resistance geneticcharacteristics of Mongolian horses. The main results showed as following:1. The results of the blood biochemical, immune and antioxidant indices showed thatthe Thoroughbred had high levels of albumin and cholesterol content in Mongolian Horse,which was consistent with the nutrition level of feed; The globulin, IgG and IgA contentsof Mongolian Horse were significantly higher than Thoroughbred (P＜0.05), whichsuggested it may be associated with the high immunity of Mongolian Horse.2. The TLR2gene was categorized into three genotypes (AA, AB and BB) byPCR-SSCP in the two Horse species. The sequencing found that there were two deletionmutations in the exon: Gâ†'C synonymous mutation at945bp and Gâ†'A missense mutation at1070bp. Arg at945bp didn’t alter due to the swing of the codon, but Arg was substitutedto Gln at1070bp. For the genotypes analysis, the distribution of genotypes had significantdifference between Mongolian Horse and Thoroughbred (0.01≤P＜0.05).3. By direct sequencing, it was found there were11base mutations in exon3of TLR4gene in both species, which included8synonymous mutations (Tâ†'G at520bp, Aâ†'G at668bp, Câ†'T at8171bp, Câ†'T at1003bp, Tâ†'C at1513bp, Gâ†'A at1546bp, Tâ†'C at1771bp, and Tâ†'C at1903bp). The left3mutation sites were: i) Câ†'A missense mutationat713bp leading to the Glnâ†'Lys amino acid substitution; ii) Tâ†'C missense mutation at1353bp leading to the Metâ†'Thr amino acid substitution; iii) Aâ†'G missense mutation at1673bp leading to the Metâ†'Val amino acid substitution. And the genotype of these3sitesshowed a significant difference between Mongolian Horse and Thoroughbred（P＜0.01).4. The differential expression of TLR1-9gene in liver, spleen, lung, caecum and bonemarrow of two species were analyzed by RT-qPCR with2-â–³Ctmethod. The results showedthat the genes mentioned above were detected in all tested tissues. The genes relativeexpression levels of Mongolian Horse were higher than Thoroughbred in immune organs(spleen and bone marrow), but lower in lung.5. In vitro treatment of equine peripheral blood monocytes with LPS increasedtranscription level of all TLR4, CD14, MD-2, TNF-α, INF-β, IL-1β, IL-6and IL-8fromboth Mongolian Horse and Thoroughbred compared with the control groups. The mRNAlevel of TLR4, CD14, IL-1β, IL-6and IL-8in Mongolian Horse was lower thanThoroughbred, and the difference of TLR4and IL-1β expression was significant (P＜0.05).The levels of the cytokines (TNF-α, INF-β, IL-1β and IL-8) in the supernatant ofperipheral blood monocytes were no difference (P≥0.05) between the horse species.6. Expression profiling libraries of Mongolian Horse-spleen and Thoroughbred-spleen were constructed, and we obtained6811804and7744386useful reads by theIllumina Miseq sequencing respectively. Most reads of two expression profiling librarieswere maped on Chromosome8and Chromosome1, and least of reads were maped onChromosome29, Chromosome30and Chromosome31.279differential genes were foundbetween two expression profiling libraries, including45immune-related genes. The GeneOntology analysis showed that differentially expressed genes were related with biologicalprocess, cellular component and molecular function. The Kyoto Encyclopedia of Genesand Genomes analysis indicated that immune system was the greatest different KEGGterm. |
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