ITRAQ-Based Quantitative Proteomic Analaysis For Identification Of Candidate Proteins Of Genetic Regulation For Horn In Sheep (Ovis Aries)

Hornless and two-horned sheep are the common phenotype in sheep breeding. Some studies have proved that the breed of multi-horned sheep exist in Europe and ancient China. The researcher constructed multi-horned sheep family and found that some horn phenotype were produced in the filial generation, like multi-horns, two horns, hornless, especially the special deformed horns. The incidence rate of the deformes horn is about 18%. The molecular etiology of sheep horn and the development pathways are still poorly known. Protein is the major carrier of performing biological functions. The differences in the number of horns and the development of deformed horn may be associated with some core proteins.In this study, we will investigate the regulatory mechanism of three horn types based on two sheep families, including Mongolia sheep and Altay sheep. We will use the basic principle of morphological structure, anatomy biopsy, X-ray, iTRAQ, Western Blotting and Q-PCR to research in these three levels——genomics, proteomics, transcriptome. The main results and conclusions were as follows:Part 1: Analysis of anatomy morphology, X-ray and tissue section. Making morphological anatomy with three phenotypes of sheep horns show that: the whole structure of normal horns are plump, its horn shape is big and symmetry. The central bone in the horn develops better than deformed horn. While the structure of deformed horn is wizened, its shape is smaller and the central born in the horn has been stopped development. It has the opposite situation. Making section with three phenotypes of sheep horns show that: no significant difference between the three phenotype horns under cross-cutting. There are some different in the deformed horn under slitting. The distance between the bone marrow in deformed horn is near. The marrow of horn is not symmetry. But the situation is opposite in the normal horn. Making photos with X-Ray for the head of different phenotype of sheep horn. It shows that: the contour of normal(two- or multi-)horn is very clear under X-Ray. Transparency of horny sheath is extremely bright and no mutation shadow to exist. In deformed horn, the contour is not clear. No development or less development can be seen in the central horn. Transparency of horny sheath is abnormal. Some fracture markers are appeared under X-Ray.Part 2: In total, 2356 proteins were screen from three phenotypes of sheep horns.1345 proteins are located in confidence area. 137 differentially expressed proteins were identified between normal two-horned group and deform-horned group. 91 differentially expressed proteins were identified between normal multi-horned group and deformed horn group. 66 differentially expressed proteins were identified between normal two-horned group and normal multi-horned group. GO enrichment analysis showed that the differentially expressed proteins in” normal-abnormal horn” group were significantly enriched in the progress of extracellular matrix organization, tissue development, collagen metabolic process. Differentially expressed proteins in” two-multi horn” group were significantly enriched in the progress of secretion by cell, secretion. Focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway may be the candidate signal pathway of deformed horn development. However, candidate signal pathway of multi-horned development is still missing found.Protein S100A9, S100A12, COL6A1, TNC, LAMB2, CHAD, PARVA, KRTAP6-1 and ACAN were proposed as key protein for deformed horn development.Part 3: Results of iTRAQ were verified by Western Blotting and Q-PCR. Expression of protein level is similar to iTRAQ. Expression of mRNA level is similar part to iTRAQ. Results of iTRAQ were believable.Part 4: Candidate genes underlying multi-horned sheep provide polymorphic loci. Five SNPs were found in MTX2 gene. Four SNPs were found in HOXD3 gene and two SNPs were found in HOXD12 gene. Two polymorphic sites may take part in the forming of multi-horn in MTX2 gene.Finally,MTX2 gene was chosen as the candidate gene for the regulation of multi-horn.